Amino polyol amine oxidase polynucleotides and related polypeptides and methods of use

ABSTRACT

The present invention provides polynucleotides and related polypeptides of the enzyme APAO isolated from  Exophiala spinifera . Additionally, the polynucleotide encoding for the APAO enzyme can be used to transform plant cells normally susceptible to Fusarium or other toxin-producing fungus infection. Plants can be regenerated from the transformed plant cells. Additionally, the present invention provides for expressing both APAO and a fumonisin esterase in a transgenic plant. In this way, a transgenic plant can be produced with the capability of degrading fumonisin, as well as with the capability of producing the degrading enzymes. In addition, the present invention provides methods for producing the APAO enzyme in both prokaryotic and non-plant eukaryotic systems. Methods for detoxification in grain, grain processing, silage, food crops and in animal feed and rumen microbes are also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/092,936 filed Jul. 15 1998 and hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates generally to the detection and isolation of fumonisin and AP1 degrading enzymes and to compositions and methods for the in vivo detoxification or degradation of fumonisin or its hydrolysis product AP1. This method has broad application in agricultural biotechnology and crop agriculture and in the improvement of food grain quality.

BACKGROUND OF THE INVENTION

Fungal diseases are common problems in crop agriculture. Many strides have been made against plant diseases as exemplified by the use of hybrid plants, pesticides and improved agricultural practices. However, as any grower or home gardener can attest, the problems of fungal plant disease continue to cause difficulties in plant cultivation. Thus, there is a continuing need for new methods and materials for solving the problems caused by fungal diseases of plants.

These problems can be met through a variety of approaches. For example, the infectious organisms can be controlled through the use of agents that are selectively biocidal for the pathogens. Another method is interference with the mechanism by which the pathogen invades the host crop plant. Yet another method, in the case of pathogens that cause crop losses, is interference with the mechanism by which the pathogen causes injury to the host crop plant. Still another method, in the case of pathogens that produce toxins that are undesirable to mammals or other animals that feed on the crop plants, is interference with toxin production, storage, or activity. This invention falls into the latter two categories.

Since their discovery and structural elucidation in 1988 (Bezuidenhout et al., Journal Chem Soc, Chem Commun 1988: 743-745 (1988)), fumonisins have been recognized as a potentially serious problem in maize-fed livestock. They are linked to several animal toxicoses including leukoencephalomalacia (Marasas, et al., Onderstepoort Journal of Veterinary Research 55: 197-204 (1988); Wilson, et al., American Association of Veterinary Laboratory Diagnosticians: Abstracts 33rd Annual Meeting, Denver, Colo., Oct. 7-9, 1990, Madison, Wis., USA) and porcine pulmonary edema (Colvin, et al., Mycopathologia 117: 79-82 (1992)). Fumonisins are also suspected carcinogens (Geary W (1971) Coord Chem Rev 7: 81; Gelderblom, et al., Carcinogenesis 12: 1247-1251 (1991); Gelderblom, et al., Carcinogenesis 13: 433-437 (1992)). Fusarium isolates in section Liseola produce fumonisins in culture at levels from 2 to >4000 ppm (Leslie, et al., Phytopathology 82: 341-345 (1992)). Isolates from maize (predominantly mating population A) are among the highest producers of fumonisin. (Leslie et al., supra). Fumonisin levels detected in field-grown maize have fluctuated widely depending on location and growing season, but both preharvest and postharvest surveys of field maize have indicated that the potential for high levels of fumonisins exists (Murphy, et al., J Agr Food Chem 41: 263-266 (1993)). Surveys of food and feed products have also detected fumonisin (Holcomb, et al., J Agr Food Chem 41: 764-767 (1993); Hopmans, et al., J Agr Food Chem 41: 1655-1658 (1993); Sydenham, et al., J Agr Food Chem 39: 2014-2018 (1991)). The etiology of Fusarium ear mold is poorly understood, although physical damage to the ear and certain environmental conditions can contribute to its occurrence (Nelson, Mycopathologia 117: 29-36 (1992)). Fusarium can be isolated from most field grown maize, even when no visible mold is present. The relationship between seedling infection and stalk and ear diseases caused by Fusarium is not clear. Genetic resistance to visible kernel mold has been identified (Gendloff, et al., Phytopathology 76: 684-688 (1986); Holley, et al., Plant Dis 73: 578-580 (1989)), but the relationship to visible mold to fumonisin production has yet to be elucidated.

Fumonisins have been shown in in vitro mammalian cell studies to inhibit sphingolipid biosynthesis through inhibition of the enzyme sphingosine N-acetyl transferase, resulting in the accumulation of the precursor sphinganine (Norred, et al., Mycopathologia 117: 73-78 (1992); Wang, et al., Biol Chem 266: 14486 (1991); Yoo, et al., Toxicol Appl Pharmacol 114: 9-15 (1992); Nelson, et al., Annu Rev Phytpathol 31:233-252 (1993)). It is likely that inhibition of this pathway accounts for at least some of fumonisin's toxicity, and support for this comes from measures of sphinganine: sphingosine ratios in animals fed purified fumonisin (Wang, et al., J Nutr 122: 1706-1716 (1992)). Fumonisins also affect plant cell growth (Abbas, et al., Weed Technol 6: 548-552 (1992); Vanasch, et al., Phytopathology 82: 1330-1332 (1992); Vesonder, et al., Arch Environ Contam Toxicol 23: 464-467 (1992)). Kuti et al., (Abstract, Annual Meeting American Phytopathological Society, Memphis, Tenn: APS Press 1993) reported on the ability of exogenously added fumonisins to accelerate disease development and increase sporulation of Fusarium moniliforme and F. oxysporum on tomato.

Enzymes that degrade the fungal toxin fumonisin to its de-esterified form (e.g. AP1 from FB1) have been identified in U.S. Pat. No. 5,716,820, issued Feb. 10, 1998 U.S. Pat. No. 5,792,931 issued Aug. 11, 1998; and pending U.S. application Ser. Nos. 08/888,950 and 08/888,949, both filed Jul. 7, 1997, and all hereby incorporated by reference. It is understood that AP1 as used here is to designate the hydrolyzed form of any fumonisin, FB1, FB2, FB3, FB4, or any other AP1-like compounds, including synthetically produced AP1 like compounds, that contain a C-2 or C-1 amine group and one or more adjacent hydroxyl groups. Plants expressing a fumonisin esterase enzyme, infected by fumonisin producing fungus, and tested for fumonisin and AP1 were found to have low levels of fumonisin but high levels of AP1. AP1 is less toxic than fumonisin to plants and probably also to animals but contamination with AP1 is still a concern. The preferred result would be complete detoxification of fumonisin to a non-toxic form. Therefore enzymes capable of degrading AP1 are necessary for the further detoxification of fumonisin. The present invention provides newly discovered polynucleotides and related polypeptides of amino polyol amine oxidase (abbreviated APAO, formerly known as AP1 catabolase, U.S. Pat. No. 5,716,820, supra; U.S. Pat. No. 5,792,931, supra, and pending U.S. applications Ser. Nos. 08/888,950 and 08/888,949, supra; trAPAO is the abbreviation for a truncated, but still functional APAO), capable of oxidatively deaminating the AP1 to a compound identified as the 2-oxo derivative of AP1 or its cyclic ketal form (abbreviated as 2-OP, formerly called AP1-N1, U.S. Pat. No. 5,716,820, supra; U.S. Pat. No. 5,792,931, supra; pending U.S. applications Ser. Nos. 08/888,950 and 08/888,949, supra), isolated from Exophiala spinifera, ATCC 74269. The partially purified APAO enzyme from Exophiala spinifera has little or no activity on intact FB1, a form of fumonisin. However, recombinant APAO enzyme from Exophiala spinifera, expressed in E. coli, has significant but reduced activity on intact FB1 and other B-series fumonisins. APAO or trAPAO thus could potentially be used without fumonisin esterase since the amine group is the major target for detoxification. Alternatively, fumoninsin esterase and APAO (or trAPAO) can be used together for degrading toxins.

APAO is a type of flavin amine oxidase (EC 1.4.3.4, enzyme class nomeclature, see Enzyme Nomenclature 1992, Recommendations of the Nomenclature Committee of the IUBMB on the Nomenclature and Classification of Enzymes, Academic Press, Inc. (1992)). Flavin amine oxidases are known in mammals as monoamine oxidases, where they participate in the conversion of amines involved in neuronal function. A prokaryotic flavin amine oxidase that deaminates putrescine has been described (Ishizuka et al., J. Gen Microbiol. 139:425-432 (1993)). A single fungal gene, from Aspergillus niger has been cloned (Schilling et al., Mol Gen Genet. 247:430-438 (1995)). It deaminates a variety of alkyl and aryl amines, but when tested for its ability to oxidize AP1, was found to not contain AP1 oxidizing activity.

The toxicity of fumonisins and their potential widespread occurrence in food and feed makes it imperative to find detoxification or elimination strategies to remove the compound from the food chain.

SUMMARY OF THE INVENTION

The present invention provides polynucleotides, related polypeptides and all conservatively modified variants of a newly discovered APAO. The nucleotide sequence of the APAO comprises the sequence found in SEQ ID NOS: 5, 10, and 22. SEQ ID NO: 5 contains the nucleotide sequence of trAPAO, SEQ ID NO: 10 contains the nucleotide sequence of trAPAO with an additional lysine and SEQ ID NO: 22 contains the full length nucleotide sequence of APAO. For expression in a plant, the nucleotide sequence of APAO or trAPAO is fused to a plant signal sequence. Preferred plant signal sequences are signal sequences which target the apoplast or a peroxisome. Other signal sequences can also be used, depending on requirements, including mitochondrial or plastidic. It is an object of the present invention to provide transgenic plants and plant cells comprising the nucleic acids of the present invention.

Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising an isolated polynucleotide sequence encoding an APAO enzyme. In a further aspect, the present invention is selected from: (a) an isolated polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide comprising at least 20 contiguous bases of the polynucleotides of the present invention; (c) a polynucleotide having at least 40% identity to a polynucleotide of the present invention; (d) a polynucleotide comprising at least 20 nucleotides in length which hybridizes under low strigency conditions to a polynucleotide of the present invention; (e) a polynucleotide comprising a polynucleotide selected from SEQ ID NOS: 5, 7, 10, 22, and 32; and (f) a polynucleotide which is complementary to the polynucleotide of (a) to (e).

Additional polynucleotides of the present invention include an APAO enzyme fused to a fumonisin esterase. The fumonisin esterase is preferably ESP1 or BEST1.

In another aspect, the present invention relates to a recombinant expression cassette comprising a nucleic acid as described, supra. Additionally, the present invention relates to a vector containing the recombinant expression cassette. Further, the vector containing the recombinant expression cassette can facilitate the transcription and translated of the nucleic acid in a host cell.

In yet another embodiment, the present invention is directed to a transgenic plant or plant cells, containing the nucleic acids of the present invention. In another embodiment, the transgenic plant is a maize plant or plant cells. In yet another embodiment are the seeds from the transgenic plant.

This invention also provides an isolated polypeptide comprising (a) a polypeptide comprising at least 25 contiguous amino acids of a polypeptide of the present invention; (b) a polypeptide comprising at least 55% sequence identity to a polypeptide of the present invention; (c) a polypeptide encoded by a nucleic acid of the present invention; (d) a polypeptide characterized by a polypeptide selected from SEQ ID NOS: 6, 11, 23, and 33; and (e) a conservatively modified variant of a polypeptide of the present invention.

Another embodiment of the subject invention comprises a method of reducing pathogenicity of a fungus producing fumonisin or a structurally related mycotoxin by transferring to a plant the nucleic acids of the present invention either by themselves or in combination with a nucleic acid coding for a fumonisin esterase. In addition, two plants, one of which is transformed with an APAO of the present invention and the other transformed with a fumonisin esterase, can be crossed to produce a plant expressing both fumonisin esterase and APAO.

This invention further provides methods of degrading a fumonisin, a fumonisin breakdown product, a structurally related mycotoxin or a breakdown product of a structurally related mycotoxin, comprising the step of reacting the mycotoxin with the degradative enzymes of the present invention. Additionally, fumonisins can be degraded by application of both fumonisin esterase enzymes and APAO enzyme. The mycotoxins can be degraded in harvested grain, during the processing of harvested grain, in animal feed, or in plant tissue as, for example, during the use of the plant for silage or as a spray on grain, fruit or vegetables.

Another embodiment of the subject invention is a host cell stably transformed by a polynucleotide construct as described above, and a method of making a polypeptide of a recombinant gene comprising:

a) providing a population of these host cells; and

b) growing the population of cells under conditions whereby the polypeptide encoded by the coding sequence of the expression cassette is expressed;

c) isolating the resulting polypeptide.

A number of expression systems using the said host cells could be used, such as but not limited to, microbial, mammalian, plant, or insect. Alternatively, the fumonisin degrading enzymes can be isolated and purified from the seeds or plant parts of a plant expressing the said enzyme.

The polynucleotides of the present invention can also be used as a selectable marker for plant transformation. By transforming plant cells with an expression cassette containing the polynucleotide of the present invention and then placing the plant cells on media containing AP1 or a phytotoxic analog, only the plant cells expressing the polynucleotide of the present invention would survive.

Another embodiment of the present invention is the use of the enzyme fumonisin esterase and APAO by themselves or in combination as reagents for detecting fumonisin and structurally related toxins.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting. The following is presented by way of illustration and is not intended to limit the scope of the invention.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., J. H. Langenheim and K. V. Thimann, Botany: Plant Biology and Its Relation to Human Affairs (1982) John Wiley; Cell Culture and Somatic Cell Genetics of Plants, Vol. 1 (I. K. Vasil, ed. 1984); R. V. Stanier, J. L. Ingraham, M. L. Wheelis, and P. R. Painter, The Microbial World, (1986) 5th Ed., Prentice-Hall; O. D. Dhringra and J. B. Sinclair, Basic Plant Pathology Methods, (1985) CRC Press; Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982); DNA Cloning, Vols. I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); and the series Methods in Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.).

Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined below are more fully defined by reference to the specification as a whole.

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

By “microbe” is meant any microorganism (including both eukaryotic and prokaryotic microorganisms), such as fungi, yeast, bacteria, actinomycetes, algae and protozoa, as well as other unicellular structures.

A “fumonisin-producing microbe” is any microbe capable of producing the mycotoxin fumonisin or analogs thereof. Such microbes are generally members of the fungal genus Fusarium, as well as recombinantly derived organisms, which have been genetically altered to enable them to produce fumonisin or analogs thereof.

By “degrading fumonisin” is meant any modification to fumonisin or AP1 molecule which causes a decrease or loss in its toxic activity, such as degradation to less than 1%, 5%, 10%, or 50% of original toxicity, with less than 10% being preferred. Such a change can comprise cleavage of any of the various bonds, oxidation, reduction, the addition or deletion of a chemical moiety, or any other change that affects the activity of the molecule. In a preferred embodiment, the modification includes hydrolysis of the ester linkage in the molecule as a first step and then oxidative deamination. Furthermore, chemically altered fumonisin can be isolated from cultures of microbes that produce an enzyme of this invention, such as growing the organisms on media containing radioactively-labeled fumonisin, tracing the label, and isolating the degraded toxin for further study. The degraded fumonisin can be compared to the active compound for its phytotoxicity or mammalian toxicity in known sensitive species, such as porcines, rabbits, and equines or in cell or tissue culture assays. Such toxicity assays are known in the art. For example, in plants a whole leaf bioassay can be used in which solutions of the active and inactive compound are applied to the leaves of sensitive plants. The leaves may be treated in situ or, alternatively, excised leaves may be used. The relative toxicity of the compounds can be estimated by grading the ensuing damage to the plant tissues and by measuring the size of lesions formed within a given time period. Other known assays can be performed at the cellular level, employing standard tissue culture methodologies e.g., using cell suspension cultures.

By “fumonisin esterase” is meant any enzyme capable of hydrolysis of the ester linkage in fumonisin or a structurally similar molecule such as AAL toxin. Two examples of such enzymes are ESP1 and BEST1 found in U.S. patent application Ser. No. 5,716,820, issued Feb. 10, 1998; U.S. Pat. No. 5,792,931 issued Aug. 11, 1998; and pending U.S. application Ser. Nos. 08/888,950 and 08/888,949, both filed Jul. 7, 1997.

By “structurally related mycotoxin” is meant any mycotoxin having a chemical structure related to a fumonisin or AP1 such as AAL toxin, fumonisin B1, fumonisin B2, fumonisin B3, fumonisin B4, fumonisin C2, fumonisin A1 and A2, and their analogs or hydrolyzed form, as well as other mycotoxins having similar chemical structures, including synthetically made analogs that contain a C-2 or C-1 amine group and one or more adjacent hydroxyl groups, that would be expected to be degraded by the activity of an enzyme of the present invention. The present invention is the first flavin amine oxidase known to attack a primary amine not located at C-1 (i.e. C-2 of AP1) and resulting in a keto rather than an aldehydic product.

It is understood that “AP1” or “amino polyol” as used here is to designate the hydrolyzed form of any fumonisin, FB1, FB2, FB3, FB4, AAL, or any other AP1-like compound, including a compound made synthetically, that contains a C-2 or C-1 amine group and one or more adjacent hydroxyl groups.

By “amplified” is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.

The term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations” and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, one exception is Micrococcus rubens, for which GTG is the methionine codon (Ishizuka, et al., J. Gen'l Microbiol, 139:425-432 (1993)) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence and incorporated herein by reference.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” when the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made. Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%, preferably 60-90% of the native protein for it's native substrate. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton (1984) Proteins W. H. Freeman and Company.

As used herein, “consisting essentially of” means the inclusion of additional sequences to an object polynucleotide where the additional sequences do not selectively hybridize, under stringent hybridization conditions, to the same cDNA as the polynucleotide and where the hybridization conditions include a wash step in 0.1×SSC and 0.1% sodium dodecly sulfate at 65° C.

By “encoding” or “encoded”, with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code. However, variants of the universal code, such as is present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum (Proc. Natl. Acad. Sci. (USA), 82: 2306-2309 (1985)), or the ciliate Macronucleus, may be used when the nucleic acid is expressed using these organisms.

When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed.

For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledonous plants or dicotyledonous plants as these preferences have been shown to differ (Murray et al. Nuel. Acids Res. 17: 477-498 (1989) and herein incorporated by reference). Thus, the maize preferred codon for a particular amino acid might be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray et al., supra.

As used herein, “heterologous” in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.

By “host cell” is meant a cell, which contains a vector and supports the replication and/or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, plant, amphibian, or mammalian cells. Preferably, host cells are monocotyledonous or dicotyledonous plant cells, including but not limited to maize, sorghum, sunflower, soybean, wheat, alfalfa, rice, cotton, canola, barley, millet, and tomato. A particularly preferred monocotyledonous host cell is a maize host cell.

The term “hybridization complex” includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.

The term “introduced” in the context of inserting a nucleic acid into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

The terms “isolated” refers to material, such as a nucleic acid or a protein, which is substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment. Nucleic acids, which are “isolated”, as defined herein, are also referred to as “heterologous” nucleic acids.

Unless otherwise stated, the term “APAO nucleic acid” means a nucleic acid comprising a polynucleotide (“APAO polynucleotide”) encoding an APAO polypeptide. The term APAO, unless otherwise stated can encompass both APAO and the functional, truncated version of APAO designated trAPAO.

As used herein, “nucleic acid” includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNA molecules, which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994 Supplement).

As used herein “operably linked” includes reference to a functional linkage between a first sequence, such as a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

As used herein, the term “plant” includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. Plant cell, as used herein includes, without limitation, seeds suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants, which can be used in the methods of the invention, is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants including species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum. A particularly preferred plant is Zea mays.

As used herein, “polynucleotide” includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

As used herein “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples are promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibres, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissue preferred”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “regulatable” promoter is a promoter, which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Another type of promoter is a developmentally regulated promoter, for example, a promoter that drives expression during pollen development. Tissue preferred, cell type specific, developmentally regulated, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter, which is active under most environmental conditions.

The term “APAO polypeptide or trAPAO polypeptide” refers to one or more amino acid sequences. The term is also inclusive of fragments, variants, homologs, alleles or precursors (e.g., preproproteins or proproteins) thereof. An “APAO or trAPAO protein” comprises an APAO or trAPAO polypeptide.

As used herein “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention. The term “recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed, and a promoter.

The term “residue” or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 40% sequence identity, preferably 60-90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) with each other.

The terms “stringent conditions” or “stringent hybridization conditions” include reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which can be up to 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Optimally, the probe is approximately 500 nucleotides in length, but can vary greatly in length from less than 500 nucleotides to equal to the entire length of the target sequence.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g, 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g, greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt's. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2× SSC (20× SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1× SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1× SSC at 60 to 65° C. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T_(m) can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984): T_(m)=81.5 ° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T_(m) is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m), hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the T_(m) can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T_(m)) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (T_(m)); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T_(m)); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)). Using the equation, hybridization and wash compositions, and desired T_(m), those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T_(m) of less than 45° C. (aqueous solution) or 32° C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, N.Y. (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, N.Y. (1995). Unless otherwise stated, in the present application high stringency is defined as hybridization in 4× SSC, 5× Denhardt's (5 g Ficoll, 5 g polyvinypyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65° C., and a wash in 0.1× SSC, 0.1% SDS at 65° C.

As used herein, “transgenic plant” includes reference to a plant, which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. “Transgenic” is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term “transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides or polypeptides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of nucleotide and amino acid sequences for comparison are well known in the art. The local homology algorithm (Best Fit) of Smith and Waterman, Adv. Appl. Math may conduct optimal alignment of sequences for comparison. 2: 482 (1981); by the homology alignment algorithm (GAP) of Needleman and Wunsch, J Mol. Biol. 48: 443 (1970); by the search for similarity method (Tfasta and Fasta) of Pearson and Lipman, Proc. Natl. Ai,ad. Sci. 85: 2444 (1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994). The preferred program to use for optimal global alignment of multiple sequences is PileUp (Feng and Doolittle, Journal of Molecular Evolution, 25:351-360 (1987) which is similar to the method described by Higgins and Sharp, CABIOS, 5:151-153 (1989) and hereby incorporated by reference). The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, N.Y. (1995).

GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters. Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).

As those of ordinary skill in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) low-complexity filters can be employed alone or in combination.

(c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences, which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences, which differ by such conservative substitutions, are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

(e) (i) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has between 50-100% sequence identity, preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of between 55-100%, preferably at least 55%, preferably at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. The degeneracy of the genetic code allows for many amino acids substitutions that lead to variety in the nucleotide sequence that code for the same amino acid, hence it is possible that the DNA sequence could code for the same polypeptide but not hybridize to each other under stringent conditions. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide, which the first nucleic acid encodes, is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

(e) (ii) The terms “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with between 55-100% sequence identity to a reference sequence preferably at least 55% sequence identity, preferably 60% preferably 70%, more preferably 80%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. In addition, a peptide can be substantially identical to a second peptide when they differ by a non-conservative change if the epitope that the antibody recognizes is substantially identical. Peptides, which are “substantially similar” share sequences as, noted above except that residue positions, which are not identical, may differ by conservative amino acid changes.

Fumonisin Degrading Organisms

The present invention is based on the discovery of organisms with the ability to degrade the mycotoxin fumonisin. In a search for a biological means of detoxifying fumonisins, several dematiaceous hyphomycetes were isolated from field-grown maize kernels. The fungi were found to be capable of growing on fumonisin B1 or B2 (FB1 or FB2) as a sole carbon source, degrading it partially or completely in the process. One species, identified as Exophiala spinifera, a “black yeast”, was recovered from maize seed from diverse locations in the southeastern and south central United States. The enzyme-active strain of Exophiala spinifera (ATCC 74269) was deposited (see U.S. patent application Ser. No. 5,716,820, issued Feb. 10, 1998; U.S. Pat. No. 5,792,931 issued Aug. 11, 1998; and pending U.S. application Ser. Nos. 08/888,950 and 08/888,949, both filed Jul. 7, 1997).

Nucleic Acids

The present invention provides, inter alia, isolated nucleic acids of RNA, DNA, and analogs and/or chimeras thereof, comprising an APAO or trAPAO polynucleotide.

The present invention also includes polynucleotides optimized for expression in different organisms. For example, for expression of the polynucleotide in a maize plant, the sequence can be altered to account for specific codon preferences and to alter GC content as according to Murray et al, supra. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra.

The APAO or trAPAO nucleic acids of the present invention comprise isolated APAO or trAPAO polynucleotides which, are inclusive of:

(a) a polynucleotide encoding an APAO or trAPAO polypeptide of the sequences found in SEQ ID NO: 6 and 22, and conservatively modified and polymorphic variants thereof;

(b) a polynucleotide which selectively hybridizes to a polynucleotide of (a) or (b);

(c) a polynucleotide having at least 40% sequence identity with polynucleotides of (a) or (b);

(d) complementary sequences of polynucleotides of (a), (b), or (c); and

(e) a polynucleotide comprising at least 15 contiguous nucleotides from a polynucleotide of (a), (b), (c), or (d).

In addition, polynucleotides are presented that are a fusion of an APAO or trAPAO polynucleotide and the polynucleotide of a fumonisin esterase. The invention encompasses the sequences from Exophiala as well as sequences having sequence similarity with such sequences. It is recognized that the sequences of the invention can be used to isolate corresponding sequences in other organisms. Methods such as PCR, hybridization, and the like can be used to identify sequences having substantial sequence similarity to the sequences of the invention. See, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Planview, N.Y.) and Innis et al., (1990) PCR Protocols: Guide to Methods and Applications (Academic Press, N.Y.). Coding sequences isolated based on their sequence identity to the entire fumonisin degrading coding sequences set forth herein or to fragments thereof are encompassed by the present invention.

It is recognized that the sequences of the invention can be used to isolate similar sequences from other fumonisin degrading organisms. Likewise sequences from other fumonisin degrading organisms may be used in combination with the sequences of the present invention. See, for example, copending application entitled “Compositions and Methods for Fumonisin Detoxification”, U.S. application Ser. No. 60/092,953, filed concurrently herewith and herein incorporated by reference.

Plasmids containing the polynucleotide sequences of the invention were deposited with American Type Culture Collection (ATCC), Manassas, Va., and assigned Accession Nos. 98812, 98813, 98814, 98815, and 98816. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. § 112.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, or combinations thereof. In some embodiments, the polynucleotides of the present invention will be cloned, amplified, or otherwise constructed from a fungus or bacteria.

The nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease,e restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention. The nucleic acid of the present invention—excluding the polynucleotide sequence—is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Typically, the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. Exemplary nucleic acids include such vectors as: M13, lambda ZAP Express, lambda ZAP II, lambda gt10, lambda gt11, pBK-CMV, pBK-RSV, pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL 4, pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS±, pSG5, pBK, pCR-Script, pET, pSPUTK, p3′SS, pGEM, pSK±, pGEX, pSPORTI and II, pOPRSVI CAT, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, pRS416, lambda MOSS1ox, and lambda MOSE1ox. Optional vectors for the present invention, include but are not limited to, lambda ZAP II, and pGEX. For a description of various nucleic acids see, for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22: 1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts. 22(20): 1859-1862 (1981), e.g., using an automated synthesizer, e.g., as described in Needham-VanDevanter et al., Nucleic Acids Res., 12: 6159-6168 (1984); and, the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill will recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

UTRs and Codon Preference

In general, translational efficiency has been found to be regulated by specific sequence elements in the 5′ non-coding or untranslated region (5′ UTR) of the RNA. Positive sequence motifs include translational initiation consensus sequences (Kozak, Nucleic Acids Res.15:8125 (1987)) and the 5<G>7 methyl GpppG RNA cap structure (Drummond et al., Nucleic Acids Res. 13:7375 (1985)). Negative elements include stable intramolecular 5′ UTR stem-loop structures (Muesing et al., Cell 48:691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5′ UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol. 8:284 (1988)). Accordingly, the present invention provides 5′ and/or 3′ UTR regions for modulation of translation of heterologous coding sequences.

Further, the polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage. Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host or to optimize the codon usage in a heterologous sequence for expression in maize. Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as “Codon Preference” available from the University of Wisconsin Genetics Computer Group (see Devereaux et al., Nucleic Acids Res. 12: 387-395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.). Thus, the present invention provides a codon usage frequency characteristic of the coding region of at least one of the polynucleotides of the present invention. The number of polynucleotides (3 nucleotides per amino acid) that can be used to determine a codon usage frequency can be any integer from 3 to the number of polynucleotides of the present invention as provided herein. Optionally, the polynucleotides will be full-length sequences. An exemplary number of sequences for statistical analysis can be at least 1, 5, 10, 20, 50, or 100.

Sequence Shuffling

The present invention provides methods for sequence shuffling using polynucleotides of the present invention, and compositions resulting therefrom. Sequence shuffling is described in PCT publication No. 96/19256. See also, Zhang, J.-H., et al. Proc. Natl. Acad. Sci. USA 94:4504-4509 (1997) and Zhao, et al., Nature Biotech 16:258-261 (1998). Generally, sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic, which can be selected or screened for. Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides , which comprise sequence regions, which have substantial sequence identity and can be homologously recombined in vitro or in vivo. The population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method. The characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property. In some embodiments, the selected characteristic will be an altered K_(m) and/or K_(cat) over the wild-type protein as provided herein. In other embodiments, a protein or polynucleotide generated from sequence shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide. In yet other embodiments, a protein or polynucleotide generated from sequence shuffling will have an altered pH optimum as compared to the non-shuffled wild-type polynucleotide. The increase in such properties can be at least 110%, 120%, 130%, 140% or greater than 150% of the wild-type value.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention. A nucleic acid sequence coding for the desired polynucleotide of the present invention, for example a cDNA or a genomic sequence encoding a polypeptide long enough to code for an active protein of the present invention, can be used to construct a recombinant expression cassette which can be introduced into the desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.

For example, plant expression vectors may include (1) a cloned plant gene under the transcriptional control of 5′ and 3′ regulatory sequences and (2) a dominant selectable marker. Such plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

A plant promoter fragment can be employed which will direct expression of a polynucleotide of the present invention in all tissues of a regenerated plant. Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the 1′- or 2′- promoter derived from T-DNA of Agrobacterium tumefaciens, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the rubisco promoter, the GRP1-8 promoter, the 35S promoter from cauliflower mosaic virus (CaMV), as described in Odell et al., (1985), Nature, 313:810-812, rice actin (McElroy et al., (1990), Plant Cell, 163-171); ubiquitin (Christensen et al., (1992), Plant Mol. Biol. 12:619-632; and Christensen, et al., (1992), Plant Mol. Biol. 18:675-689); pEMU (Last, et al., (1991), Theor. Appl. Genet. 81:581-588); MAS (Velten et al., (1984), EMBO J 3:2723-2730); and maize H3 histone (Lepetit et al., (1992), Mol. Gen. Genet. 231:276-285; and Atanassvoa et al., (1992), Plant Journal 2(3):291-300), ALS promoter, as described in published PCT Application WO 96/30530, and other transcription initiation regions from various plant genes known to those of skill. For the present invention ubiquitin is the preferred promoter for expression in monocot plants.

Alternatively, the plant promoter can direct expression of a polynucleotide of the present invention in a specific tissue or may be otherwise under more precise environmental or developmental control. Such promoters are referred to here as “inducible” promoters. Environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light. Examples of inducible promoters are the Adh1 promoter, which is inducible by hypoxia or cold stress, the Hsp70promoter, which is inducible by heat stress, and the PPDK promoter, which is inducible by light.

Examples of promoters under developmental control include promoters that initiate transcription only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds, or flowers. The operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.

If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region can be derived from a variety of plant genes, or from T-DNA. The 3′ end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene. Examples of such regulatory elements include, but are not limited to, 3′ termination and/or polyadenylation regions such as those of the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan et al., (1983), Nucl. Acids Res. 12:369-385); the potato proteinase inhibitor II (PINII) gene (Keil, et al., (1986), Nucl. Acids Res. 14:5641-5650; and An et al., (1989), Plant Cell 1:115-122); and the CaMV 19S gene (Mogen et al., (1990), Plant Cell 2:1261-1272).

An intron sequence can be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. Cell Biol. 8: 4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

Plant signal sequences, including, but not limited to, signal-peptide encoding DNA/RNA sequences which target proteins to the extracellular matrix of the plant cell (Dratewka-Kos, et al., (1989), J. Biol. Chem. 264:4896-4900), the Nicotiana plumbaginifolia extension gene (DeLoose, et al., (1991), Gene 99:95-100), signal peptides which target proteins to the vacuole like the sweet potato sporamin gene (Matsuka, et al., (1991), PNAS 88:834) and the barley lectin gene (Wilkins, et al., (1990), Plant Cell, 2:301-313), signal peptides which cause proteins to be secreted such as that of PRIb (Lind, et al., (1992), Plant Mol. Biol. 18:47-53), or the barley alpha amylase (BAA) (Rahmatullah, et al., Plant Mol. Biol. 12:119 (1989)) and hereby incorporated by reference), or from the present invention the signal peptide from the ESP1 or BEST1 gene, or signal peptides which target proteins to the plastids such as that of rapeseed enoyl-Acp reductase (Verwaert, et al., (1994), Plant Mol. Biol. 26:189-202) are useful in the invention. The barley alpha amylase signal sequence fused to the trAPAO polynucleotide (see SEQ ID NO: 20) is the preferred construct for expression in maize for the present invention.

The vector comprising the sequences from a polynucleotide of the present invention will typically comprise a marker gene, which confers a selectable phenotype on plant cells. Usually, the selectable marker gene will encode antibiotic resistance, with suitable genes including genes coding for resistance to the antibiotic spectinomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance, genes coding for resistance to herbicides which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, and the ALS gene encodes resistance to the herbicide chlorsulfuron.

Alternatively, the invention, itself, could be used as a method for selection of transformants, in other words as a selectable marker. An APAO or trAPAO polynucleotide operably linked to a promoter and then transformed into a plant cell by any of the methods described in the present application would express the degradative enzyme. When the plant cells are placed in the presence of either AP1 or a phytotoxic analog in culture only the transformed cells would be able to grow. In another embodiment, the plant cell could be transformed with both a polynucleotide for APAO and a polynucleotide for fumonisin esterase. The selective agent in this case could be either AP1 or fumonisin or any structural analog. Thus, growth of plant cells in the presence of a mycotoxin favors the survival of plant cells that have been transformed to express the coding sequence that codes for one of the enzymes of this invention and degrades the toxin. When the APAO or trAPAO cassette with or without the fumonisin esterase polynucleotide, is co-transformed with another gene of interest and then placed in the presence of fumonisin, AP1 or a phytotoxic analog, this invention would allow for selection of only those plant cells that contain the gene of interest. In the past antibiotic resistance genes have been used as selectable markers. Given the current concerns by consumers and environmentalist over use of antibiotic genes and the possibility of resistant microorganisms arising due to this use, a non-antibiotic resistant selectable marker system such as the present invention, fulfills this very important need.

Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al., Meth. In Enzymol., 153:253-277 (1987). These vectors are plant integrating vectors in that on transformation, the vectors integrate a portion of vector DNA into the genome of the host plant. Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl et al., Gene, 61:1 -11 (1987) and Berger et al., Proc. Natl. Acad. Sci. U.S.A., 86:8402-8406 (1989). Another useful vector herein is plasmid pBI101.2 that is available from CLONTECH Laboratories, Inc. (Palo Alto, Calif.).

Expression of Proteins in Host Cells

Using the nucleic acids of the present invention, one may express a protein of the present invention in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian, or preferably plant cells. The cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention. To obtain high level expression of a cloned gene, it is desirable to construct expression vectors which contain, at the minimum, a strong promoter, such as ubiquitin, to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator. Constitutive promoters are classified as providing for a range of constitutive expression. Thus, some are weak constitutive promoters, and others are strong constitutive promoters. Generally, by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By “low level” is intended at levels of about 1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Conversely, a “strong promoter” drives expression of a coding sequence at a “high level”, or about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts.

One of skill would recognize that modifications could be made to a protein of the present invention without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.

A. Expression in Prokaryotes

Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., Nature 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake et al., Nature 292:128 (1981)). The inclusion of selection markers in DNA vectors transfected in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.

The vector is selected to allow introduction of the gene of interest into the appropriate host cell. Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al., Gene 22: 229-235 (1983); Mosbach, et al., Nature 302: 543-545 (1983)). The pGEX-4T-1 plasmid vector from Pharmacia is the preferred E. coli expression vector for the present invention.

B. Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, the present invention can be expressed in these eukaryotic systems. In some embodiments, transformed/transfected plant cells, as discussed infra, are employed as expression systems for production of the proteins of the instant invention.

Synthesis of heterologous proteins in yeast is well known. Sherman, F., et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well recognized work describing the various methods available to produce the protein in yeast. Two widely utilized yeasts for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.

A protein of the present invention, once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates or the pellets. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.

The sequences encoding proteins of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. A number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol. Rev. 89: 49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7th edition, 1992).

Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth, and Drosophila cell lines such as a Schneider cell line (See Schneider, J. Fmbryol. Exp. Morphol. 27: 353-365 (1987).

As with yeast, when higher animal or plant host cells are employed, polyadenlyation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45: 773-781 (1983)). Additionally, gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors. Saveria-Campo, M., Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA Cloning Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press, Arlington, Va. pp. 213-238 (1985).

In addition, one of the genes for fumonisin esterase or the APAO or trAPAO placed in the appropriate plant expression vector can be used to transform plant cells. The enzyme can then be isolated from plant callus or the transformed cells can be used to regenerate transgenic plants. Such transgenic plants can be harvested, and the appropriate tissues (seed or leaves, for example) can be subjected to large scale protein extraction and purification techniques, and the fumonisin degradation enzymes or APAO can be isolated for use in fumonisin and flumonisin hydrolysis product detoxification processes.

Plant Transformation Methods

Numerous methods for introducing foreign genes into plants are known and can be used to insert an APAO or trAPAO polynucleotide into a plant host, including biological and physical plant transformation protocols. See, for example, Miki et al., (1993), “Procedure for Introducing Foreign DNA into Plants”, In: Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pages 67-88. The methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, microorganism-mediated gene transfer such as Agrobacterium (Horsch, et al., (1985), Science 227:1229-31), electroporation, micro-injection, and biolistic bombardment.

Expression cassettes and vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are known and available. See, for example, Gruber, et al., (1993), “Vectors for Plant Transformation” In: Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds. CRC Press, Inc., Boca Raton, pages 89-119.

Agrobacterium-mediated Transformation

The most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria, which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of plants. See, for example, Kado, (1991), Crit. Rev. Plant Sci. 10:1. Descriptions of the Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided in Gruber et al., supra; Miki, et al., supra; and Moloney et al., (1989), Plant Cell Reports 8:238.

Similarly, the gene can be inserted into the T-DNA region of a Ti or Ri plasmid derived from A. tumefaciens or A. rhizogenes, respectively. Thus, expression cassettes can be constructed as above, using these plasmids. Many control sequences are known which when coupled to a heterologous coding sequence and transformed into a host organism show fidelity in gene expression with respect to tissue/organ specificity of the original coding sequence. See, e.g., Benfey, P. N., and Chua, N. H. (1989) Science 244: 174-181. Particularly suitable control sequences for use in these plasmids are promoters for constitutive leaf-specific expression of the gene in the various target plants. Other useful control sequences include a promoter and terminator from the nopaline synthase gene (NOS). The NOS promoter and terminator are present in the plasmid pARC2, available from the American Type Culture Collection and designated ATCC 67238. If such a system is used, the virulence (vir) gene from either the Ti or Ri plasmid must also be present, either along with the T-DNA portion, or via a binary system where the vir gene is present on a separate vector. Such systems, vectors for use therein, and methods of transforming plant cells are described in U.S. Pat. No. 4,658,082; U.S. application Ser. No. 913,914, filed Oct. 1, 1986, as referenced in U.S. Pat. No. 5,262,306, issued Nov. 16, 1993 to Robeson, et al.; and Simpson, R. B., et al. (1986) Plant Mol. Biol. 6: 403-415 (also referenced in the '306 patent); all incorporated by reference in their entirety.

Once constructed, these plasmids can be placed into A. rhizogenes or A. tumefaciens and these vectors used to transform cells of plant species, which are ordinarily susceptible to Fusarium or Alternaria infection. Several other transgenic plants are also contemplated by the present invention including but not limited to soybean, corn, sorghum, alfalfa, rice, clover, cabbage, banana, coffee, celery, tobacco, cowpea, cotton, melon and pepper. The selection of either A. tumefaciens or A. rhizogenes will depend on the plant being transformed thereby. In general A. tumefaciens is the preferred organism for transformation. Most dicotyledonous plants, some gymnosperms, and a few monocotyledonous plants (e.g. certain members of the Liliales and Arales) are susceptible to infection with A. tumefaciens. A. rhizogenes also has a wide host range, embracing most dicots and some gymnosperms, which includes members of the Leguminosae, Compositae, and Chenopodiaceae. Monocot plants can now be transformed with some success. European Patent Application Publication Number 604 662 A1 to Hiei et al. discloses a method for transforming monocots using Agrobacterium. Saito et al. discloses a method for transforming monocots with Agrobacterium using the scutellum of immature embryos (European Application 672 752 A1). Ishida et al. discusses a method for transforming maize by exposing immature embryos to A. tumefaciens (Ishida et al, Nature Biotechnology, 1996, 14:745-750).

Once transformed, these cells can be used to regenerate transgenic plants, capable of degrading fumonisin. For example, whole plants can be infected with these vectors by wounding the plant and then introducing the vector into the wound site. Any part of the plant can be wounded, including leaves, stems and roots. Alternatively, plant tissue, in the form of an explant, such as cotyledonary tissue or leaf disks, can be inoculated with these vectors, and cultured under conditions, which promote plant regeneration. Roots or shoots transformed by inoculation of plant tissue with A. rhizogenes or A. tumefaciens, containing the gene coding for the fumonisin degradation enzyme, can be used as a source of plant tissue to regenerate fumonisin-resistant transgenic plants, either via somatic embryogenesis or organogenesis. Examples of such methods for regenerating plant tissue are disclosed in Shahin, E. A. (1985) Theor. Appl. Genet. 69:235-240; U.S. Pat. No. 4,658,082; Simpson, R. B., et al. (1986) Plant Mol. Biol. 6: 403-415; and U.S. patent applications Ser. Nos. 913,913 and 913,914, both filed Oct. 1, 1986, as referenced in U.S. Pat. No. 5,262,306, issued Nov. 16, 1993 to Robeson, et al.; the entire disclosures therein incorporated herein by reference.

Direct Gene Transfer

Despite the fact that the host range for Agrobacterium-mediated transformation is broad, some major cereal crop species and gymnosperms have generally been recalcitrant to this mode of gene transfer, even though some success has recently been achieved in rice (Hiei et al., (1994), The Plant Journal 6:271-282). Several methods of plant transformation, collectively referred to as direct gene transfer, have been developed as an alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation is microprojectile-mediated transformation, where DNA is carried on the surface of microprojectiles measuring about 1 to 4 μm. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate the plant cell walls and membranes. (Sanford et al., (1987), Part. Sci. Technol. 5:27; Sanford, 1988, Trends Biotech 6:299; Sanford, (1990), Physiol. Plant 79:206; Klein et al., (1992), Biotechnology 10:268).

Another method for physical delivery of DNA to plants is sonication of target cells as described in Zang et al., (1991), BioTechnology 9:996. Alternatively, liposome or spheroplast fusions have been used to introduce expression vectors into plants. See, for example, Deshayes et al., (1985), EMBO J. 4:2731; and Christou et al., (1987), PNAS USA 84:3962. Direct uptake of DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol, or poly-L-ornithine has also been reported. See, for example, Hain et al., (1985), i Mol. Gen. Genet. 199:161; and Draper et al., (1982), Plant Cell Physiol. 23:451.

Electroporation of protoplasts and whole cells and tissues has also been described. See, for example, Donn et al., (1990), In: Abstracts of the VIIth Int'l. Congress on Plant Cell and Tissue Culture IAPTC, A2-38, page 53; D'Halluin et al., (1992), Plant Cell 4:1495-1505; and Spencer et al., (1994), Plant Mol. Biol. 24:51-61.

Thus, polynucleotide encoding a polypeptide able to inactivate fumonisin or AP1 can be isolated and cloned in an appropriate vector and inserted into an organism normally sensitive to the Fusarium or its toxins. Furthermore, the polynucleotide imparting fumonisin or AP1 degradative activity can be transferred into a suitable plasmid, and transformed into a plant. Thus, a fumonisin or AP1 degrading transgenic plant can be produced. Organisms expressing the polynucleotide can be easily identified by their ability to degrade fumonisin or AP1. The protein capable of degrading fumonisin or AP1 can be isolated and characterized using techniques well known in the art.

APAO or trAPAO in a Transgenic Plant

Fumonisin esterase reduces but does not eliminate the toxicity of fumonisins. Therefore a second enzymatic modification to further reduce or abolish toxicity is desirable. The partially purified APAO enzyme from Exophiala spinifera has little or no activity on intact FB1, a form of fumonisin. However, recombinant APAO enzyme from Exophiala spinifera, expressed in E. coli, has significant but reduced activity on intact FB1 and other B-series fumonisins. APAO or trAPAO thus could potentially be used without fumonisin esterase since the amine group is the major target for detoxification. Alternatively, the two genes, fumoninsin esterase and APAO (or trAPAO) can be used together for degrading toxins.

APAO is predicted to be an enzyme that, when by itself or co-expressed in a heterologous expression system along with fumonisin esterase (either ESP1 or BEST1), will result in the production of 2-oxo pentol (2-OP) from fumonisin B1. The substrate range of recombinant, E. coli-expressed APAO is limited to fumonisins and their hydrolysis products and does not include amino acids, sphingolipid precursors such as phytosphingosine, or polyamines such as spermidine. Thus, APAO is highly specific for fumonisin-like amines, and thus would have little deleterious effect on other cellular metabolites. In addition, if it is extracellularly localized, it will limit any contact with biologically important amines that might also be substrates. The end result will be a more effective detoxification of fumonisins than can be achieved with esterase alone.

The oxidase activity of APAO is predicted to result in generation of hydrogen peroxide in stoichiometric amounts relative to AP1 or fumonisin oxidized. This may prove to be an additional benefit of this enzyme, since hydrogen peroxide is both antimicrobial and is thought to contribute to the onset of a defense response in plants (Przemylaw, Biochem J, 322:681-692 (1997), Lamb, et al, Ann Rev Plant Physiol Plant Mol Bio 48:251-275 (1997), and Alverez, et al., Oxidative Stress and the Molecular Biology of Antioxidant Defenses, Cold Spring Harbor Press, 815-839 (1997)).

Since one of the preferred embodiments of the present invention is to have both a fumonisin esterase polynucleotide and an APAO or trAPAO polynucleotide present in a plant, there are several ways to introduce more than one polynucleotide in a plant. One way is to transform plant tissue with polynucleotides to both fumonisin esterase and APAO or trAPAO at the same time. In some tissue culture systems it is possible to transform callus with one polynucleotide and then after establishing a stable culture line containing the first polynucleotide, transform the callus a second time with the second polynucleotide. One could also transform plant tissue with one polynucleotide, regenerate whole plants, then transform the second polynucleotide into plant tissue and regenerate whole plants. The final step would then be to cross a plant containing the first polynucleotide with a plant containing the second polynucleotide and select for progeny containing both polynucleotides.

Another method is to create a fusion protein between esterase and APAO or trAPAO, preferably with a spacer region between the two polypeptides. Both enzymes would be active although tethered to each other. In addition, an enzyme cleavage site engineered in the spacer region, would allow cleavage by an endogenous or introduced protease. Transgenic plants containing both a fumonisin esterase enzyme and/or the APAO enzyme and thus able to degrade fumonisin or a structurally related mycotoxin would be able to reduce or eliminate the pathogenicity of any microorganism that uses fumonisin or a structurally related mycotoxin as a mode of entry to infect a plant. Fungal pathogens frequently use toxins to damage plants and weaken cell integrity in order to gain entry and expand infection in a plant. By preventing the damage induced by a toxin, a plant would be able to prevent the establishment of the pathogen and thereby become tolerant or resistant to the pathogen.

Another benefit of fumonisin degradation is the production of hydrogen peroxide. When fumonisin is broken down to 2-OP, hydrogen peroxide is produced as a by-product. Hydrogen peroxide production can trigger enhanced resistance responses in a number of ways. 1) Hydrogen peroxide has direct antimicrobial activity. 2) Hydrogen peroxide acts as a substrate for peroxidases associated with lignin polymerization and hence cell wall strengthening. 3) Via still to be determined mechanisms, hydrogen peroxide acts as a signal for activation of expression of defense related genes, including those that result in stimulation of salicylic acid accumulation. Salicylic acid is thought to act an endogenous signal molecule that triggers expression of genes coding for several classes of pathogenesis-related proteins. Moreover, salicylic acid may set up the oxidative burst and thus act in a feedback loop enhancing its own synthesis. Salicylic acid may also be involved in hypersensitive cell death by acting as an inhibitor of catalase, an enzyme that removes hydrogen peroxide. 4) Hydrogen peroxide may trigger production of additional defense compounds such as phytoalexins, antimicrobial low molecular weight compounds. For a review on the role of the oxidative burst and salicylic acid please see Lamb, C. and Dixon, R. A., Ann. Rev. Plant Physiol. Plant Mol. Biol., 48: 251-275 (1997).

Detoxification of Harvested Grain, Silage, or Contaminated Food Crop

The present invention also relates to a method of detoxifying a fumonisin or a structurally related mycotoxin with the APAO enzyme from by Exophiala spinifera, ATCC 74269 during the processing of grain for animal or human food consumption, during the processing of plant material for silage, or food crops contaminated with a toxin producing microbe, such as but not limited to, tomato. Since the atmospheric ammoniation of corn has proven to be an ineffective method of detoxification (see B. Fitch Haumann, INFORM 6:248-257 (1995)), such a methodology during processing is particularly critical where transgenic detoxification is not applicable.

In one embodiment of the present invention, fumonisin degradative enzymes are presented to grain, plant material for silage, or a contaminated food crop, or during the processing procedure, at the appropriate stages of the procedure and in amounts effective for detoxification of fumonisins and structurally related mycotoxins. Detoxification by the enzymes, the microbial strains, or an engineered microorganism can occur not only during the processing, but also any time prior or during the feeding of the grain or plant material to an animal or incorporation of the grain or food crop into a human food product, or before or during ingestion of the food crop.

Another embodiment of the present invention is the engineering of a bacterium or fungus to express the detoxification enzymes and then using the bacterium or fungus rather than the enzyme itself. There are a number of microbes that could be engineered to express the polynucleotides of the present invention. One could also activate, either inducibly or constitutively, the endogenous genes for fumonisin esterase or APAO of Exophiala. By overexpressing the degradative enzymes and then treating plants, seed, or silage with the microorganism, it would be possible to degrade fumonisin in situ.

The polynucleotides of the invention can be introduced into microorganisms that multiply on plants (epiphytes) to deliver enzymes to potential target crops. Epiphytes can be gram-positive or gram-negative bacteria, for example.

The microorganisms that have been genetically altered to contain at least one degradative polynucleotide and resulting polypeptide may be used for protecting agricultural crops and products. In one aspect of the invention, whole, i.e. unlysed, cells of the transformed organism are treated with reagents that prolong the activity of the enzyme produced in the cell when the cell is applied to the environment of a target plant. A secretion leader may be used in combination with the gene of interest such that the resulting enzyme is secreted outside the host cell for presentation to the target plant.

The degradative enzymes can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray. Any suitable microorganism can be used for this purpose. See, for example, Gaertner, et al. (1993) in Advanced Engineered Pesticides, (ed. Kim, Marcel Dekker, N.Y.).

The enzymes or microorganisms can be introduced during processing in appropriate manners, for example as a wash or spray, or in dried or lyophilized form or powered form, depending upon the nature of the milling process and/or the stage of processing at which the enzymatic treatment is carried out. See generally, Hoseney, R. C., Principles of Cereal Science and Technology, American Assn. of Cereal Chemists, Inc., 1990 (especially Chapters 5, 6 and 7); Jones, J. M., Food Safety, Eagan Press, St. Paul, Minn., 1992 (especially Chapters 7 and 9); and Jelen, P., Introduction to Food Processing, Restan Publ. Co., Reston, Va., 1985. Processed grain or silage to be used for animal feed can be treated with an effective amount of the enzymes in the form of an inoculant or probiotic additive, for example, or in any form recognized by those skilled in the art for use in animal feed. The enzymes of the present invention are expected to be particularly useful in detoxification during processing and/or in animal feed prior to its use, since the enzymes display relatively broad ranges of pH activity. The esterase from Exophiala spinifera, ATCC 74269, showed a range of activity from about pH 3 to about pH 6, and the esterase from the bacterium of ATCC 55552 showed a range of activity from about pH 6 to about pH 9 (U.S. Pat. No. 5,716,820, supra). The APAO enzyme from Exophiala spinifera (ATCC 74269) has a pH range of activity from pH 6 to pH 9.

Genetic Engineering of Ruminant Microorganisms

Ruminant microorganisms can be genetically engineered to contain and express either the fumonisin esterase enzymes or APAO isolated from Exophiala spinifera, ATCC 74269, or a combination of the enzymes. The genetic engineering of microorganisms is now an art recognized technique, and ruminant microorganisms so engineered can be added to feed in any art recognized manner, for example as a probiotic or inoculant. In addition, microorganisms capable of functioning as bioreactors can be engineered so as to be capable of mass producing either the fumonisin esterases or the APAO enzyme found in Exophiala spinifera, ATCC 74269.

Use of the Fumonisin Esterase and APAO Enzymes for Detection of Reagents for Fumonisins and Related Compounds

Another embodiment of the present invention is the use of the enzymes of the present invention as detection reagents for fumonisins and related compounds. The enzymes of the present invention can be used as detection reagents because of the high specificity of the esterase and deaminase enzymes, and the fact that hydrolysis followed by amine oxidation can be monitored by detection of hydrogen peroxide or ammonia using standard reagents (analogous to a glucose detection assay using glucose oxidase). Hydrogen peroxide is often measured by linking a hydrogen peroxide-dependent peroxidase reaction to a colored or otherwise detectable peroxidase product (e.g. Demmano, et al., European Journal of Biochemistry 238(3): 785-789 (1996)). Ammonia can be measured using ion-specific electrodes: Fritsche, et al., Analytica Chimica Acta 244(2): 179-182 (1991); West, et al., Analytical Chemistry 64(5): 533-540 (1992), and all herein incorporated by reference) or by GC or other chromatographic method.

For example, recombinant or non-recombinant, active fumonisin esterase (ESP1 or BEST) and APAO proteins are added in catalytic amounts to a sample tube containing an unknown amount of fumonisins (FB1, FB2, FB3, FB4, or partial or complete hydrolysis products of these). The tube is incubated under pH and temperature conditions sufficient to convert any fumonisin in the sample to AP1, and correspondingly the AP1 to 2-OP, ammonia, and hydrogen peroxide. Then suitable reagents are added for quantification of the hydrogen peroxide or ammonia that were generated stoichiometrically from fumonisins. By comparison with control tubes that received no esterase or APAO enzyme, the amount of fumonisin present can be calculated in direct molar proportion to the hydrogen peroxide or ammonia detected, relative to a standard curve.

This invention can be better understood by reference to the following non-limiting examples. It will be appreciated by those skilled in the art that other embodiments of the invention may be practiced without departing from the spirit and the scope of the invention as herein disclosed and claimed.

EXAMPLE 1

Fungal and Bacterial Isolates

Exophiala isolates from maize were isolated as described in U.S. Pat. No. 5,716,820, issued Feb. 10, 1998 and pending U.S. applications Ser. Nos. 08/888,950 and 08/888,949, both filed Jul. 7, 1997, and herein incorporated by reference.

Isolation Methods

Direct isolation of black yeasts from seed was accomplished by plating 100 microliters of seed wash fluid onto YPD or Sabouraud agar augmented with cycloheximide (500 mg/liter) and chloramphenicol (50 mg/liter). Plates were incubated at room temperature for 7-14 days, and individual pigmented colonies that arose were counted and cultured for analysis of fumonisin-degrading ability as described in U.S. Pat. No. 5,716,820, issued Feb. 10, 1998 and pending U.S. application Ser. Nos. 08/888,950 and 08/888,949, both filed Jul. 7, 1997.

Analysis of Fumonisins and Metabolism Products

Analytical thin-layer chromatography was carried out on 100% silanized C18 silica plates (Sigma #T-7020; 10×10 cm; 0.1 mm thick) by a modification of the published method of Rottinghaus (Rottinghaus, et al., J Vet Diagn Invest, 4: 326 (1992), and herein incorporated by reference).

To analyze fumonisin esterase activity sample lanes were pre-wet with methanol to facilitate sample application. After application of from 0.1 to 2 μl of aqueous sample, the plates were air-dried and developed in MeOH:4% KCl (3:2) or MeOH:0.2 M KOH (3:2) and then sprayed successively with 0.1 M sodium borate (pH 9.5) and fluorescamine (0.4 mg/ml in acetonitrile). Plates were air-dried and viewed under long wave UV.

For analysis of APAO activity, an alternative method was used. Equal volumes of sample and ¹⁴C-AP1 (1 mg/ml, pH 8, 50 mM sodium phosphate) were incubated at room temperature for one to six days. Analytical thin-layer chromatography was then carried out on C60 HPK silica gel plates (Whatman #4807-700; 10×10 cm; 0.2 mm thick). After application of from 0.1 to 2 μl of aqueous sample, the plates were air-dried and developed in CHCl₃:MeOH:CH₃COOH:H₂O (55:36:8:1). Plates were then air dried, and exposed to Phosphorlmager screen (Molecular Dynamics) or autoradiographic film. A Storm™ Phosphorlmager (Molecular Dynamics) was used to scan the image produced on the screen.

Alkaline Hydrolysis of FB1 to AP1

FB1 or crude fumonisin C₈ material was suspended in water at 10-100 mg/ml and added to an equal volume of 4 N NaOH in a screw-cap tube. The tube was sealed and incubated at 60° C. for 1 hr. The hydrolysate was cooled to RT and mixed with an equal volume of ethyl acetate, centrifuged at 1000 RCF for 5 minute and the organic (upper) layer recovered. The pooled ethyl acetate layers from two successive extractions were dried under N₂ and resuspended in distilled H₂O. The resulting material (the aminopentol of FB1 or “AP1”) was analyzed by TLC.

Enzyme Activity of Culture Filtrate and Mycelium

Exophiala spinifera isolate 2141.10 was grown on YPD agar for 1 week, and conidia were harvested, suspended in sterile water, and used at 105 conidia per ml to inoculate sterile Fries mineral salts medium containing 1 mg/ml purified FB1 (Sigma Chemical Co.). After 2 weeks incubation at 28° C. in the dark, cultures were filtered through 0.45 micron cellulose acetate filters, and rinsed with Fries mineral salts. Fungal mycelium was suspended in 15 mL of 0.1% FB1, pH 5.2+1 mM EDTA+3 μg/mL Pepstatin A+1.5 μg/mL Leupeptin and disrupted in a Bead Beater™ using 0.1 mm beads and one minute pulses, with ice cooling. Hyphal pieces were collected by filtering through Spin X™ (0.22 μm), and both mycelial supernatant and original culture filtrates were assayed for fumonisin modification by methods outlined above.

Preparation of Crude Culture Filtrate

Agar cultures grown as above were used to inoculate YPD broth cultures (500 ml) in conical flasks at a final concentration of 105 conidia per ml culture. Cultures were incubated 5 days at 28° C. without agitation and mycelia harvested by filtration through 0.45 micron filters under vacuum. The filtrate was discarded and the mycelial mat was washed and resuspended in sterile carbon-free, low mineral salts medium (1 g/liter NH₃NO₄; 1 g/liter NaH₂PO₄; 0.5 g/liter MgCl₂; 0.1 g/liter NaCl; 0.13 g/liter CaCl₂; 0.02 g/liter FeSO₄.7H₂O, pH 4.5) containing 0.5 mg/ml alkaline hydrolyzed crude FB1. After 3-5 days at 28° C. in the dark with no agitation the cultures were filtered through low protein binding 0.45 micron filters to recover the culture filtrate. Phenylmethyl sulfonyl fluoride (PMSF) was added to a concentration of 2.5 mM and the culture filtrate was concentrated using an Amicon™ YM10 membrane in a stirred cell at room temperature, and resuspended in 50 mM sodium acetate, pH 5.2 containing 10 mM CaCl₂. The crude culture filtrate (approx. 200-fold concentrated) was stored at −20° C.

To obtain preparative amounts of enzyme-hydrolyzed fumonisin, 10 mg. of FB1 (Sigma) was dissolved in 20 mL of 50 mM sodium acetate at pH 5.2+10 mM CaCl₂, and 0.25 mL of 200×concentrated crude culture filtrate of 2141.10 was added. The solution was incubated at 37° C. for 14 hours, and then cooled to room temperature. The reaction mixture was brought to approx. pH 9.5 by addition of 0.4 mL of 4 N KOH, and the mixture was extracted twice with 10 mL ethyl acetate. The combined organic layers were dried under N₂ and resuspended in dH₂O. 2.5 milligrams of organic extracted material were analyzed by Fast Atom Bombardment (FAB) mass spectrometry. The resulting mass spectrum showed a major ion at M/z (+1)=406 mass units, indicating the major product of enzymatic hydrolysis was AP1which has a calculated molecular weight of 405.

EXAMPLE 2 Preparation of AP1-induced and Non-induced Mycelium

Liquid cultures of Exophiala spinifera isolate 2141.10 were prepared from YPD agar plates (Yeast Extract 10 gm, Bacto-Peptone 20 gm, Dextrose 0.5 gm, and Bacto-Agar 15 gm per liter of water). Aliquots (400-500 uL) of a water suspension of E. spinifera cells from YPD agar were spread uniformly onto 150×15 mm YPD agar plates with 4 mm sterile glass beads. The plates were incubated at room temperature for 6-7 days. The mycelia/conidia were transferred from the agar plates into Mineral Salts Medium (MSM) (Na₂HPO₄7H₂O 0.2 gm, NH₄Cl 1.0 gm, CaCl₂2H₂O 0.01 gm, FeSO₄7H₂O 0.02 gm per liter of distilled water, pH 4.5) and centrifuged at 5000× g, 4° C., 20 minutes to pellet the cells. The cell pellet was rinsed once in 40 mL MSM and recentrifuged. The rinsed cell pellet was used to inoculate MSM at a 1:19 ratio of packed cells: MSM. The culture to be induced was supplemented with AP1 to a final concentration of 0.5-1.0 mg/ml and incubated at 28° C., 100 rpm, in the dark to induce catabolic enzymes. The non-induced cultures did not receive AP1 but were place on media containing 4-ABA at the same concentration as AP1. The supernatants were removed by filtration through 0.45 cellulose acetate. The remaining mycelial mat was washed with sterile MSM and then frozen in liquid nitrogen for storage.

EXAMPLE 3 Effect of FB1 and AP1on Maize Coleoptiles

Maize coleoptiles from 4 day dark-grown germinated maize seeds were excised above the growing point and placed in 96-well microtiter plates in the presence of 60 microliters of sterile distilled water containing FB1 or AP1 at approximately equimolar concentrations of 1.5, .5, .15, .05, .015, .005, .0015, or .0005 millimolar, along with water controls. After 2 days in the dark at 28° C. the coleoptiles were placed in the light and incubated another 3 days. Injury or lack thereof was evaluated as follows:

0 .0005 .0015 .005 .015 .05 .15 .5 1.5 mM FB1 − − − − +/− + + + + AP1 − − − − − − − − + + = brown necrotic discoloration of coleoptile − = no symptoms (same as water control)

The results indicate there is at least a 30-fold difference in toxicity between FB1 and AP1 to maize coleoptiles of this genotype. This is in general agreement with other studies where the toxicity of the two compounds was compared for plant tissues: In Lemna tissues, AP1 was approx. 40-fold less toxic (Vesonder et al.,” Arch Environ Contam Toxicol 23: 464-467 (1992).). Studies with both AAL toxin and FB1 in tomato also indicate the hydrolyzed version of the molecule is much less toxic (Gilchrist et al., Mycopathologia 117: 57-64 (1992)). Lamprecht et al. also observed an approximate 100-fold reduction in toxicity to tomato by AP1 versus FB1 (Lamprecht et al., Phytopathology 84:383391(1994)).

EXAMPLE 4 Effect of FB1 and AP1 on Maize Tissue Cultured Cells (Black Mexican Sweet, BMS)

FB1 or AP1 at various concentrations was added to suspensions of BMS cells growing in liquid culture medium in 96-well polystyrene plates. After 1 week the cell density in wells was observed under low power magnification and growth of toxin-treated wells was compared to control wells that received water. Growth of BMS cells was significantly inhibited at 0.4 micromolar FB1, but no inhibition was observed until 40 micromolar AP 1. This represents an approximate 100-fold difference in toxicity to maize tissue cultured cells. Similarly Van Asch et al. (VanAsch et al., Phytopathology 82: 1330-1332 (1992)) observed significant inhibition of maize callus grown on solid medium at 1.4 micromolar FB 1. AP1 was not tested in that study, however.

EXAMPLE 5 APAO Activity

A cell-free extract that contains the amine oxidase activity was obtained by subjecting substrate-induced Exophiala spinifera cells to disruption using a Bead Beater™ in 50 mM Na-phosphate, pH 8.0, and recovering the cell-free supernatant by centrifugation and 0.45 micron filtration. Catabolic activity is assayed by incubating extracts with AP1 (hydrolyzed fumonisin B1 backbone) or ¹⁴C-labelled AP1 with the extract and evaluating by TLC on C18 or C60 silica. The product 2-OP has a lower Rf than AP 1 and is detected either by radiolabel scan or by H₂S0 ₄ spray/charring of the TLC plate. 2-OP does not react with the amine reagent fluorescamine that is routinely used to detect AP1 on TLC plates, suggesting that the amine group is missing or chemically modified. Activity is greater at 37° C. than at room temperature, but following 30 min. at 65° C. or 100° C. no AP1 catabolic activity remained. Activity is maximal at pH 9. At pH 9, complete conversion to 2-OP occurred in 30 minutes. Activity is retained by 30,000 dalton molecular weight cutoff membrane, but only partially retained by 100,000 dalton molecular weight cutoff membrane. Other amine-containing substrates were tested for modification by the crude extract. Fumonisin (with tricarballylic acids attached) is not modified by the extract, indicating that ester-hydrolysis must occur first for the APAO to be able to be effective in modifying FB 1. Other long-chain bases (sphingosine, sphinganine, and phytosphingosine) are apparently not modified by the crude APAO, suggesting the enzyme(s) is specific for the fumonisin backbone. Preparative amounts of the product, named 2-OP, have also been purified and analyzed by C13 nmr. The results indicate that 2-OP has a keto group at carbon 2 instead of an amine, consistent with an oxidative deamination by an amine oxidase. The C13 -nmr data also indicate that 2-OP spontaneously forms an internal hemiketal between C-1 and C-5, resulting in a 5-membered ring with a new chiral center at C-2. All other carbon assignments are as in AP1, thus 2-OP is a compound of composition C₂₂H₄₄O₆, FW 404. The product of the enzyme acting on hydrolyzed fumonisin would not be expected to display any significant toxicity.

Other enzymes were tested for their ability to modify AP1. All enzymes were assayed by radiolabeled TLC, as described above, under optimal conditions at 37° Celsius, overnight or longer. The results are as follows:

Deaminating EC Source Result Monoamine Oxidase 1.4.3.4 bovine plasma negative D-amino oxidase 1.4.3.3 porcine kidney; TypeX negative L-amino oxidase 1.4.3.2 C. adamanteus venom; negative TypeI Tyramine oxidase 1.4.3.4 Arthrobacter spp negative Methylamine 1.4.99.3 Paracoccus denitrificans negative dehydrogenase Aralkyl amine 1.4.99.4 Alcaligenes faecalis negative dehydrogenase Phenylalanine 4.3.1.5 Rhodotorula glutinis; TypeI negative ammonia lyase Histidine 4.3.1.3 Pseudomonas fluorescens negative ammonia lyase L-aspartase 4.3.1.1 Hafnia alvei negative (Bacterium cadaveris) Tyrosine oxidase 1.14.18.1 mushroom negative Lysine oxidase 1.4.3.14 Trichoderma viride negative Diamine oxidase 1.4.3.6 porcine kidney negative

The results were negative for each enzyme tested. Therefore isolates from the American Type Culture Collection (ATCC) were collected. The ATCC isolates selected were listed as containing amine-modifying enzymes or were capable of growth/utilization on amine-containing substrates. The isolates were tested to determine if they could grow on or utilize AP1 as the sole nitrogen source and if any could modify AP1 to a new compound(s). The nitrogen sources that were used in liquid cultures were AP1 0.1% (w/v), s-butylamine 0.1% (v/v), n-butylamine 0.1% (v/v), and ammonium nitrate 0.2% (w/v). These were prepared in Vogel's Minimal Media (without NH₄NO₃) containing 2% sucrose. The isolates were inoculated into the various media and monitored for growth over 2-3 weeks. They were also assayed with the ¹⁴C-radiolabeled TLC assay for AP1 modification. In summary, none of the isolates tested exhibited modification of AP1 in vivo. Clearly the APAO enzyme from Exophiala spinifera is unique and unusual in its ability to modify the AP1 toxin.

EXAMPLE 6 Isolation of the trAPAO Polynucleotide

The trAPAO polynucleotide was identified using a proprietary transcript imaging method that compares transcript patterns in two samples and allows cloning of differentially expressed fragments. This technology was developed by CuraGen® (New Haven, Conn.). (see Published PCT patent application No. WO 97/15690, published May 1, 1997, and hereby incorporated by reference) Fluorescently-tagged, PCR amplified cDNA fragments representing expressed transcripts can be visualized as bands or peaks on a gel tracing, and the cDNA from differentially expressed (induced or suppressed) bands can be recovered from a duplicate gel, cloned and sequenced. Known cDNAs can be identified without the need for cloning, by matching the predicted size and partially known sequence of specific bands on the tracing.

In the present invention two RNA samples were obtained from cultures of E. spinifera grown for a specified period in a mineral salts medium containing either AP1 (induced condition), or gamma-aminobutyric acid (ABA; non-induced condition) as a sole carbon source. In the induced condition, fumonisin esterase and APAO enzyme activities are detected, whereas in the non-induced condition these activities are not detected. The methods used for induction of APAO and detection of activity are described earlier (see Example 2 and Example 5). RNA was extracted from induced mycelium by Tri-Reagent methods (Molecular Research Center Inc., Cincinnati, Ohio) only grinding a frozen slurry of tissue and Tri-Reagent with a mortar and pestle until almost melted and adding an additional extraction after the phase separation by extracting the aqueous phase one time with phenol, and two times with a phenol:chloroform:isoamyl alcohol mixture. The RNA's were submitted for CuraGeng® transcript imaging to detect cDNA fragments that are induced specifically in the presence AP1. In the resulting gel tracing several bands were found which showed induction of at least 2-fold up to 79-fold or even 100-fold or more in AP1. In the resulting gel tracing several bands were found which showed induction of at least 10-fold in AP1-grown cells as compared to cells grown in ABA. The sequence of two highly induced bands can be found in Table 1.

TABLE 1 Nucleotide sequence of two CuraGen ® bands that were identified as strongly induced by AP1 in cultures of Exophiala spinifera. >k0n0-395.5_b (SEQ ID NO: 1) GGGCCCCGGCGTTCTCGTAGGCTGCGCGGAGTTGGTCCCAGACAGACTTTTGTCGTACCTGCTTG GACTGTTGGGACCACTTCCGTCCCGGGTCTCCGACCATGAAACAGGTAATGGACCATTGTCGAT CGACGTCGATGCTGGTATCTCTGGCAAATGAGATGGGGTCACAGCTCGATTGGAGGACGCCCGA GAAGCCTTGTTCGCGCCACCACGGCTTGTCCCATACGAAGACTATCTTGCTATAGTAGCCCAGG ATAGAATTTTCCGCCAATGCTTGCTTCTCGGCGGGAAGAGGTGGTGAAAATGTCAAGGTGGGAT ACAAGGTTGTCGGTAACGAAACCANCACCTTTTTGCTTCGGAACACGGCGC > r0c0-182.3_6(SEQ ID NO: 2) GAATTTTCCGCCAATGCTTGCTTCTCGGCGGGAAGAGGTGGTGAAAATGTCAAGGTGGGATACA AGGTTGTCGGTAACGAAACCACCACCTTTTTGCTTCGGAACACGGCGCCCGAGGCCGATCGTAC TGTACAGCCGGATGCCGACTGCTCAATTTCAGCGACGGGGGTGTTGAGGTGCAC

Two of the highly induced bands, k0n0-395.5, and r0c0-182.3 showed significant sequence homology to a family of enzymes, flavin-containing amine oxidases (EC 1.4.3.4), that oxidize primary amines to an aldehyde or ketone, releasing ammonia and hydrogen peroxide (Table 2).

TABLE 2 Identification of a putative flavin amine oxidase from E. spinifera: AP1-induced transcript fragments with amine oxidase homology. BLAST 2.0 default parameters. Clone Best Best Hit Name, Likely ID Size Hit source Prob from to function k0n0- 395 bp P40974 putrescine oxidase, 8.0 e 276 333 oxidation of 395.5 Micrococcus rubens, -07 C-2 amine of EC 1.4.3.10 AP1 Length = 478 r0c0- 182 bp P12398 monoamine oxidase type 0.0039 238 296 oxidation of 182.3 A (MAO-A) [Bos taurus] C-2 amine of (contigs Length = 527 AP1 with k0n0- 395)

The chemical structure of the primary product of AP1 deamination is thought to be a 2-keto compound which cyclizes to a hemiketal at carbons 2 and 5. Therefore it is predicted that this induced enzyme is responsible for deamination of AP1.

Using sequence derived from k0n0-395.5, a partial cDNA was obtained by 3′ and 5′ RACE-PCR (Chenchik, et al., CLONTECHniques X 1:5-8 (1995); Chenchik, et al., A new method for full-length cDNA cloning by PCR. In A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis. Ed. Krieg, P. A. (Wiley-Liss, Inc.), 273-321 (1996)). A RACE cloning kit from CLONTECH was used, to obtain the RACE amplicons. Briefly, poly A+ RNA is transcribed to make first strand cDNA using a “lock-docking” poly T, cDNA synthesis primer, the second strand is synthesized and the Marathon cDNA adaptor is ligated to both ends of the ds cDNA. Diluted template is then used with the Marathon adapter primer and in separate reactions either a 5′ Gene Specific Primer (GSP) or a 3′GSP is used to produce the 3′ or 5′ RACE amplicon. After characterization of the RACE product(s) and sequencing, full-length cDNAs may be generated by 1) end-to-end PCR using distal 5′ and 3′ GSPs with the adapter-ligated ds cDNA as template, or 2) the cloned 5′ and 3′-RACE fragments may be digested with a restriction enzyme that cuts uniquely in the region of overlap, the fragments isolated and ligated. Subsequently, the RACE-generated full-length cDNAs from 1) and 2) may be cloned into a suitable vector.

In combination with the supplied adapter primer the following gene specific primers were used: for 3′RACE the oligonucleotide N21965: 5′-TGGTTTCGTTACCGACAACCTTGTATCCC-3′ (SEQ ID NO: 3) and for 5′ RACE, the oligonucleotide N21968: 5′-GAGTTGGTCCCAGACAGACTTTTGTCGT-3′ (SEQ ID NO: 4). The nucleotide sequence of the trAPAO polynucleotide, k0n0-395_(—)6.5, from Exophiala spinifera is shown in SEQ ID NO: 5. The polypeptide sequence of trAPAO is shown in SEQ ID NO: 6.

A second clone of APAO containing an unspliced intron was also found. The polynucleotide sequence of trAPAO-I polynucleotide, k0n0-395_(—)5.4, the intron containing clone, from Exophiala spinifera, can be found in SEQ ID NO: 7. The polypeptide sequence of trAPAO-I with the intron spliced out is shown in SEQ ID NO: 8. The polypeptide sequence of trAPAO-I without the intron spliced out is shown in SEQ ID NO: 9.

EXAMPLE 7 Heterologous Expression of trAPAO

Protein alignments generated with PileUp (GCG) indicate that k0n0-395_(—)6.5 (trAPAO) is similar in size to other flavin amine oxidases and is close to being full length with respect to the amino terminus of their class of proteins. The k0n0-395_(—)6.5 sequence contains a complete , β-α-βfold that is required for dinucleotide (FAD) binding, close to the amino end. The k0n0-395 sequence appears to lack only a variable amino terminal segment that varies in length from 5 amino acids in rat monoamine oxidases A & B to 40 amino acids in length in Aspergillus MAO-N. The function of these amino terminal extensions is not known; they are not recognizable as secretion signals. Based on the likely localization of the Exophiala APAO outside the cell membrane, the prediction is that k0n0-395 would have a signal sequence similar to that of the fumonisin esterase cloned from the same organism (U.S. Pat. No. 5,716,820, supra). Using GenomeWalker™, it is possible to clone the 5′ end of the transcript and upstream genomic regulatory elements. However, the signal sequence is not expected to be critical to the functionality of the enzyme; in fact, the preferred strategy for heterologous expression in maize and Pichia pastoris involves replacing the endogenous signal sequence (if present) with an optimized signal sequence for the organism, e.g. barley alpha amylase for maize and the yeast alpha factor secretion signal for Pichia. In maize transformed with fumonisin esterase, the barley alpha amylase signal sequence gave higher amounts of functional protein than the native fungal signal, therefore replacement of the native fungal signal sequence is a logical optimization step. Since many of the amine oxidases have a positively charged amino acid near the N-terminus and upstream of the dinucleotide binding site, an additional optimization step included adding a codon for the lysine (K) to the N-terminus of the trAPAO clone (k0n0-395_(—)6.5, SEQ ID NO: 5). This clone is designated K:trAPAO and can be seen in SEQ ID NOS: 10 and 11. The extra lysine is at amino acid 1 and nucleotides 1-3.

EXAMPLE 8 Pichia Expression of trAPAO

For optimum expression of trAPAO in Pichia pastoris the alpha mating factor signal peptide was fused in-frame with K:trAPAO coding sequence and can be seen in SEQ ID NOS: 16 and 17. The nucleotide sequence of clone pPicZalphaA:K:trAPAO contains a PCR-amplified insert comprising the k0n0-395 open reading frame with an additional lysine residue at the amino terminus, with a 5′ EcoRI site and 3′ NotI site for in-frame cloning into the alpha factor secretion vector pPicZalphaA. Nucleotides 1-267 contain the yeast α mating factor secretion signal. The amino acid sequence of shown in SEQ ID NO: 17 contains the trAPAO polypeptide produced from pPicZalphaA:K:trAPAO following transformation into Pichia pastoris.

For cloning into the Pichia pastoris expression vector, two cloning strategies were used. The cDNA k0n0-395_(—)5.4 was generated by using end-to-end PCR using distal 5′ and 3′ GSPs with the adapter-ligated ds cDNA as template. pPicZalphaA, distal oligonucleotide primers were designed with 5′ restriction enzyme sites that contain a 23-25 bp anchored overlap of the 5′ end (sense strand) and 3′ end (antisense strand) for cloning into the open reading frame of k0n0-395; the 3′ primer also included the stop codon. The primer sequences are N23256: 5′-ggggaattcAAAGACAACGTTGCGGACGTGGTAG-3′ (SEQ ID NO: 12) and N23259: 5′-ggggcggccgcCTATGCTGCTGGCACCAGGCTAG-3′ (SEQ ID NO: 13). A second method was used to generate k0n0-395_(—)6.5. 5′ RACE and 3′ RACE products were generated using a distal primer containing the necessary restriction enzyme sites, stop codon, etc as described above and paired with a “medial” GSP. The “medial primers” N21965: 5′-TGGTTTCGTTACCGACAACCTTGTATCCC-3′ (SEQ ID NO: 14) for 3′ RACE and for 5′ race, the oligonucleotide N21968: 5′-GAGTTGGTCCCAGACAGACTTTTGTCGT-3′ (SEQ ID NO: 15). Adapter-ligated ds cDNA was used as template. The isolated 5′ and 3′-RACE fragments were digested with a restriction enzyme that cuts uniquely in the region of overlap, in this case Bgl I, isolated and ligated into the expression vector. The digestible restriction sites allow cloning of the inserts in-frame into EcoRI/NotI digested pPicZalphaA. pPicZalphaA is an E. coli compatible Pichia expression vector containing a functional yeast alpha factor secretion signal and peptide processing sites, allowing high efficiency, inducible secretion into the culture medium of Pichia. The resulting 1.4 kb bands were cloned into EcoRI/NotI digested pPicZalphaA plasmid.

SEQ ID NO: 16 contains the polynucleotide sequence of clone pPicZalphaA:K:trAPAO, a PCR-amplified insert that comprises the k0n0-395 open reading frame with an additional lysine residue at the amino terminus, and a 5′ EcoRI site and 3′ NotI site for in-frame cloning into the alpha factor secretion vector pPicZalphaA. SEQ ID NO: 17 contains the amino acid sequence of the trAPAO polypeptide produced from pPicZalphaA:K:trAPAO following transformation into Pichia pastoris. The alpha factor secretion signal and a lysine are added.

Pichia was transformed as described in Invitrogen Manual, Easy Select™ Pichia Expression Kit, Version B, #161219, with the trAPAO polynucleotide as described above with either an intron (trAPAO-I, negative control, no expression of active trAPAO since Pichia does not splice introns very efficiently) or without an intron (capable of making an active APAO protein). The Pichia culture fluids and pellets were assayed for APAO activity as described earlier.

The set of frozen six day Pichia culture cell pellets contained two samples with intron (SEQ ID NO: 7) in gene construct, # 11, # 14, and two samples without intron in gene construct (SEQ ID NO: 5), #6, # 52. The six day culture fluids from the same cultures were used to spike with crude fungal enzyme for positive controls.

The 50 μl cell pellets were resuspended in 150 μl cold 50 mM Na-phosphate, pH 8.0, and divided into two fresh 500 μl tubes. One tube was kept on ice with no treatment, the pellet suspension, and one tube was used for lysis. An equal volume of 0.1 mm zirconia-silica beads was added to each tube. The tubes were BeadBeat™ for 15 seconds then cooled on ice 5 minutes. This was repeated three times. The crude lysate was then transferred to another tube for assay or lysate suspension.

The TLC assays were performed as follows, the samples are 1) pellet suspensions; 10 μl; 2) lysate suspensions; 10 1μl; 3) media controls-mixed 5 μl media with 5 1μl crude fungal enzyme; 10 μl; 4) positive control-used crude fungal enzyme undiluted; 10 μl; 5) substrate control-used 50 mM Na-phosphate, pH8.0; 10 μl. Ten microliters of each sample plus 10 μl of ¹⁴C-AP1 (1 mg/ml, 50 mM Na-phosphate, pH 8) was incubated at room temperature for 6 days. One microliter of the sample was spotted onto C18 and C60 TLC plates. The C18 plates were developed in MeOH:4% KCl (3:2). The C60 plastes were developed in CHCl₃:MeOH:CH₃COOH:H₂O (55:36:8:1). The plates were then air dried and then exposed to a PhosphorScreen™ for 2-3 days. A Storm™ Phosphorlmager was used to develop the images.

A positive TLC result is obtained if an additional radioactive spot appears at a lower Rf of the produced AP1 modification earlier identified as 2-OP, a deaminated product of AP1. In samples # 6 and # 52 (without intron) the AP1-modifying enzyme activity (conversion of AP1 to 2-OP) was detected in pellet suspensions and pellet lysates, although the majority of activity was associated with the pellet suspensions. In samples #11 and #14 (with intron) a minimal amount of AP1-modifying enzyme activity was detectable in the pellet lysate of # 14 only, which indicates Pichia cannot process the intron efficiently.

This experiment verified APAO activity can be detected in Pichia transformants, which verifies that trAPAO as described functions correctly in degrading AP1. The activity is associated with cell suspensions, which show higher activity than pellet lysates. Pellet lysates may show less activity due to release of endogenous proteases during lysis of the cells.

EXAMPLE 9 Expression of trAPAO in E. coli

The vector for expressing K:trAPAO in E. coli is pGEX-4T-1. This vector is a prokaryotic glutathione S-transferase (GST) fusion vector for inducible, high-level intracellular expression of genes or gene fragments as fusions with Schistosoma japonicum GST. GST gene fusion vectors include the following features, a lac promoter for inducible, high-level expression; an internal lac Iq gene for use in any E coli host; and the thrombin factor Xa or PreScission Protease recognition sites for cleaving the desired protein from the fusion product. The insert of interest, k0n0-395_(—)6.5 (K:trAPAO), was subcloned into the 5′ EcoRI site and a 3′ NotI site allowing in-frame expression of the GST:K:trAPAO fusion peptide. The polynucleotide sequence of the GST:K:trAPAO fusion can be found in SEQ ID NO: 18. The GST fusion with polylinker can be found at nucleotides 1 to 687. The K:trAPAO can be found at nucleotides 688 to 2076. The resulting polypeptide for the GST:K:trAPAO fusion can be seen at SEQ ID NO: 19. Amino acids 1 to 229 represent the GST fusion plus polyliker and amino acids 230 to 692 represent the K:trAPAO portion of the fusion.

E. coli was transformed with the pGEX-4T-1 vector containing K:trAPAO as described in BRL catalogue, Life Technologies, Inc. catalogue; Hanahan, D., J. Mol. Biol. 166:557 (1983) Jessee, J. Focus 6:4 (1984); King, P. V. and Blakesley, R., Focus 8:1, 1 (1986), and hereby incorporated by reference. The transformed E. coli was induced by addition of IPTG (isopropyl b-D-thiogalactopyranoside). Four samples of soluble extract and four samples of insoluble inclusion bodies were tested for trAPAO activity as described in Example 9. APAO activity was present in all soluble samples and two insoluble samples. Highest activity was found at 10 uM IPTG induction. Thus the pGEX-4T-1 vector containing k0n0-395_(—)6.5 construct is capable of producing active APAO enzyme in E. coli.

EXAMPLE 10 The Complete Nucleotide Sequence of the Exophiala APAO Gene

Using Genome Walker, the complete nucleotide sequence of the Exophiala APAO gene was recovered. The nucleotide sequence described in SEQ ID NO: 5 is missing a portion of the 5′ end of the native gene. The missing portion of the 5′ end of the native gene is not necessary for expression of an active APAO enzyme, as can be seen in Examples 9 and 10. The complete nucleotide sequence of APAO can be seen in SEQ ID NO: 22. The translation of SEQ ID NO: 22 can be found in SEQ ID NO: 23.

EXAMPLE 11 Expression of APAO and ESP1 in Transgenic Maize Callus

One of the preferred constructs for expression in maize is the nucleotide sequence of the trAPAO fused to the barley alpha amylase signal sequence. The nucleotide sequence of K:trAPAO translational fusion with barley alpha amylase signal sequence, for expression and secretion of the mature trAPAO in maize can be seen in SEQ ID NO: 20. Nucleotides 1-72, represent the barley alpha amylase signal sequence; nucleotides 73-75, represent the added lysine residue; and nucleotides 76-1464, represent the trAPAO cDNA. The amino acid sequence translation of SEQ ID NO: 20 can be found in SEQ ID NO: 21. Amino acids 1 to 24 represent the barley alpha amylase signal sequence and amino acids 25 to 463 is the sequence of K:trAPAO.

Maize embryos were transformed with linear DNA (insert, lacking a bacterial antibiotic resistance marker), derived from constructs containing three transcription units: 1) a PAT selectable marker gene (Wohlleben et al., Gene 70, 25-37 (1988)), 2) flumonisin esterase ESP1 fused to a barley alpha amylase signal sequence, and 3) full length APAO without or with an amino-terminal barley alpha amylase signal sequence, (P13603, comprising a PAT selectable marker fused to a 35S promoter, fumonisin esterase ESP1 fused to a barley alpha amylase signal sequence and the ubiquitin promoter, and APAO fused to the ubiquitin promoter and P13611, comprising a PAT selectable marker fused to the 35S promoter, fumonisin esterase ESP1fused to a barley alpha amylase signal sequence and the ubiquitin promoter and APAO fused to a barley alpha amylase signal sequence and the ubiquitin promoter). In these constructs both ESP1 and APAO were linked to the maize ubiquitin promoter and first intron. In a third construct, the same three transcriptional units were cloned into an Agrobacterium T1 vector (P15258, the construct comprises a PAT selectable marker, fumonisin esterase ESP1 fused to a barley alpha amylase signal sequence and APAO). Stably transformed callus or T0 plants regenerated from callas were tested for ESP 1 and APAO activity in buffer extracts of leaf tissue, using radiolabeled FB1 and/or AP1 and C18 thin-layer chromatography. Positive controls consist of non-transformed tissue spiked with E coli-expressed recombinant ESP1 or APAO. The results indicate that both ESP1 and APAO activities can be detected in transgenic maize callus and plants.

Expression of ESP1 and APAO in Transgenic Callus

Sample ID Construct Number ESP1 activity (TLC) APAO activity (TLC) 13603 3065.031-2 + + 13603 3065.034-3 + + 13603 3065.1117-3 + + 13603 3065.11s7-n13 + + 13603 3065.117-2 + + 13603 3065.1115-2 + + 13603 3065.1115-6 + + 13603 3065.1112-1 + + 13603 3065.118-6 + + 13603 3065.11s3-1 + + 13603 3065.11s1-13 + + 13603 2805.762-2 + + 13603 3065.1110-2 + + 13603 3065.039-2 + + 13611 3065.293-3 + + 13611 3065.263-1 + + 13611 3070.24.2.3 + +

Transgenic plants were regenerated from the transgenic callus positive for both ESP1 and APAO activity by standard methods known in the art. Enzyme activity was tested as described previously. As can be seen below transgenic maize plants can successfully express both ESP1 and APAO enzymes.

Expression of APAO and ESP1 in Transgenic Maize Plants (T0)

Sample Construct ID Number ESP1 activity (TLC) APAO activity (TLC) 13603 910080 + + 13603 910081 + + 13603 917065 + +

Another preferred construct for expression of APAO in a plant is targeting the APAO to the peroxisome. Maize embryos were bombarded with insert containing APAO operably linked to ubiquitin promoter and a peroxisomal targeting sequence (Gould, et al., J Cell Biol 108:1657-1664 (1989)); ESP1 operably linked to ubiquitin promoter and the barley alpha amylase signal sequence; and a selectable marker of PAT operably linked to the 35S promoter (construct number 114952). Negative controls were unbombarded embryos/callus. Positive controls were unbombarded embryos/callus spiked with purified enzyme. Transformed callus was then tested for ESP1 or APAO activity as previously described. Out of 67 samples tested 18 samples contained both ESP1 activity and APAO activity. Peroxisomally targeted APAO and apoplast targeted fumonisin esterase can both be successfully expressed in a plant cell.

Another preferred construct for expression of APAO in a plant is targeting the APAO to the mitochondrial membrane. A C-terminal extension is required for targeting monoamine oxidases MAO-A and MAO-B to mammalian outer mitochondrial membranes. An MAO-A, MAO-B, or functionally similar C-terminal extension can be fused in-frame to APAO or trAPAO to facilitate localization of this enzyme to the mitochondrial membrane of maize or other transformed species.

EXAMPLE 12 Comparison of APAO Sequence With Other Sequences

The Exophiala cDNA of APAO (SEQ ID NO: 22) contains an 1800 bp open reading frame coding for a 600 amino acid polypeptide (SEQ ID NO: 23) with divergent homology to two classes of proteins. The carboxy three-fourths of APAO (amino acids 137 to 593) is strongly homologous to flavin amine oxidases, a group of enzymes catalyzing the oxidative deamination of primary amines at carbon 1. The amine oxidase function of the carboxy terminal domain was confirmed by expression of a truncated APAO polypeptide (from 137 to 600) in both Pichia pastoris and E. coli, using AP1 as a substrate (see Example 9). The amino terminal portion of APAO, in contrast, (from approx. 5 to 134) shows significant homology to a group of small deduced open reading frames (ORFs) reported in several bacteria and blue-green algae, as well as several higher organisms. These ORFs code for small proteins of unknown function, ranging in size from 14 to 17 kDA. The juxtaposition of these divergent homologies in a single polypeptide has not been reported previously.

Flavin amine oxidases (E.C. 4.1.4.3) are a group of flavoenzymes found in both higher and lower organisms, and serve a variety of functions in catabolism. They catalyze the oxidative deamination of primary amino groups located at the C-1 position of a variety of substrates, resulting in an aldehyde product plus ammonia and hydrogen peroxide. The APAO enzyme described in this report is the first flavin amine oxidase known to attack a primary amine not located at C-1 (i.e. C-2 of AP1) and resulting in a keto rather than aldehydic product. IHowever, amino acid oxidases, while not closely related to flavin amine oxidases, are flavoenzymes that oxidize a C-2 amine adjacent to a C-1 carboxyl group.

The monoamine oxidases MAO A & B, (from human, bovine, and trout), are localized in the mitochondrial outer membrane of higher organisms and regulate the level of neurotransmitters. Microbial examples include a fungal amine oxidase (Aspergillus niger (niger) MAO-N) involved in amine catabolism, and a bacterial putrescine oxidase from a gram (+) bacterium (Micrococcus rubens.). The primary polypeptides vary in length from 478 to 527 amino acids, and share regions of high amino acid sequence conservation at the 5′ end as well as at various points through the coding region. Protein alignments generated with PileUp (GCG) indicate that trAPAO contains all conserved domains found in this class of proteins including those near the 5′ end.

The amine oxidase domain of trAPAO contains several key features shared by this class of enzymes, including an amino-terminal dinucleotide (ADP) binding region characterized by a beta-alpha-beta stretch containing three invariant glycines (G-X-G-X-X-G) in the beta-alpha turn. In trAPAO, this sequence is (DVVVVGAGLSG). This region is involved in FAD binding. Absent are several features unique to the mammalian amine oxidases, including several essential cysteine residues (Wu et al., Mol Pharm 43:888 (1993)), one of which (Cys-406 of MAO-A) is involved in covalent binding of FAD, and a carboxy-terminal extension that has been demonstrated to be involved in transporting to and anchoring the MAO in the outer mitochondrial membrane. The Aspergillus enzyme MAO-N has been demonstrated to contain non-covalent FAD, and also lacks the conserved cysteine. Therefore it is possible that the Exophiala APAO enzyme has a non-covalent FAD. The Aspergillus MAO-N has a carboxy-terminal tripeptide Ala-Arg-Leu that is involved in peroxisomal targeting and localization; this, sequence is absent from Exophiala APAO.

The amine oxidase domain of trAPAO contains a total of seven cysteines, compared to ten for the Aspergillus enzyme and only two for the Micrococcus enzyme. The mammalian MAO enzymes contain variable numbers of cysteines (at least ten), some of which are highly conserved (including the FAD binding residue mentioned above). The trAPAO sequence also has two putative glycosylation sites (NDS, NQS) towards the amino end.

The purpose of the amino-terminal extension of APAO and the basis for its homology to a group of 14-17 kDa proteins is not clear. In Synechocystis, a similar polypeptide ORF is located immediately upstream of the NADP-dependent glutamine dehydrogenase (gdhA) and has been shown to be required for functional expression of gdhA (Chavez et al, 1995). However, in trAPAO the domain is clearly not necessary for enzymatic activity, as shown by the results of the expression experiments using the truncated APAO. An interesting clue comes from the frequent association of this small ORF with gene clusters involved in oxidoreductase activity in bacteria, or induced by heat stress in mice, suggesting a possible role in redox protection. A byproduct of amine oxidase activity is hydrogen peroxide. Flavoenzymes and other redox enzymes are often susceptible to inactivation by hydrogen peroxide (Schrader et al., App Microb Biotechnol 45:458; Aguiree, et al., J Bacteriol 171:6243 (1989)), and it is possible that this protein has a protective role against oxidants such as hydrogen peroxide. Alternatively, this domain could be involved in enzyme function, localization or association of the enzyme with other structures. No signal peptide region can be detected in this amino terminal region.

In multiple sequence alignment using GCG PileUp, trAPAO is most similar to putrescine oxidase of Micrococcus rubens, Swissprot accession number P40974, (30% identical amino acids, 40% similar). Homology with several mammalian monoamine oxidases A and B, Swissprot accession numbers P21397 (Homo Sapiens mao a), P19643 (Rattus norvegicus mao b), P21396 (Rattus norvegicus mao a), and P21398 (Bos taurus mao a), is somewhat less, ranging from 25 to 28% identity and 36 to 40% similarity. Homology to the only other fungal flavin amine oxidase known, MAO-N from Aspergillus niger (Swissprot accession number P46882), is somewhat lower (24% identical, 34% similar). The microbial enzymes are considerably divergent from each other, while the mammalian monoamine oxidases share 65 to 87% identity.

The amino terminal domain (ATD) of APAO also shows homology to a 14.5 kD protein from human and rat phagocytes that shows translational inhibition activity in vitro (Swissprot accession # P52758, P52759) Schmiedeknecht, et al., Eur J Biochem 242 (2), 339-351 (1996)), and includes a heat-responsive protein from mouse (Samuel, et al., Hepatology 25 (5), 1213-1222 (1997)). This suggests that this family of proteins is involved in regulating cellular metabolism. No example exists in which this domain is fused to a larger protein domain, however, making APAO unique. Without intending to be limited by theory, all of this suggests, that this domain plays a regulatory role in APAO gene expression, possibly to prevent translation of the message when it is not needed. This raises the question of how translation of the message is restored when active enzyme is required by the Exophiala cell. Possibly there are alternative start sites that begin downstream of the inhibitor domain; or proteolysis, complexing, degradation, or phosphorylation/dephosphorylation of the inhibitor domain when it is not needed. The first possibility is less likely because there are no other ATG codons prior to the ATG at 122-124 that constitutes the predicted start site of APAO. The second possibility cannot be easily tested, although there is a casein kinase site in the ATD. Alternative roles for the ATD include oligomerization of the APAO protein, or anchoring the protein to some intracellular site, such as the membrane.

A parallel example of regulatory control over another flavoenzyme, human flavin monooxygenase 4 (FMO-4), by a C-terminal extention has been reported (Itagaki, et al., J of Biol Chem 271(33): 20102-20107 (1996)). In this case the introduction of a stop codon prior to the 81 base C-terminal extension allowed expression of active enzyme in heterologous systems. The role of the C-terminal portion was not elucidated, however. In another example, alternative splicing led to a shorter gene product that complexed with and interfered with the function of the normally spliced version (Quinet, et al., J of Biol Chem 268(23): 16891-16894 (1993)). In another case, an alternative splicing-generated insert in another protein led to inhibition of cell growth (Bhat, et al., Protein Engineering 9(8): 713-718 (1996)). In yet another variation, fas/Apol splicing variants prevent apoptosis, apparently through a 49 amino acid domain shared by all variants ((Papoff, et al., J of Immunology 156(12): 4622-4630 (1996)).

EXAMPLE 13 Making a Fusion Protein Containing Fumonisin Esterase and AP Amine Oxidase Activity in the Same Polypeptide

The enzyme activities of fumonisin esterase and AP amine oxidase can be combined in a single polypeptide by using the open reading frames together either with or without a spacer region between the two polypeptides. This creates a hybrid protein with dual enzyme activities that can be exported as a unit to the apoplast, and will allow both enzyme activities to be conveniently localized to the same area of the cell wall. The two cDNA's can be combined in either order, but the preferred method is to link them in the order NH₃-Esterase:Amine Oxidase-COOH. The spacer, if present, may consist of a short stretch of amino acids such as GGGSGGGS, or a set of amino acids that comprises a protease cleavage site that can be acted on by an apoplastic protease. This would result in the production of stoichiometric amounts of both esterase and APAO enzymes in the apoplast.

The esterase-APAO fusion protein can be made with either the fumonisin esterase from E. spinifera 2141.19 (ESP1) or fumonisin esterase from bacterium 2412.1 (BEST1). Since the pH range for maximum activity of BEST1 is similar to that of APAO (range 6.0 to 8.0), these may present the most effective combination in fusion form. As described in previous examples these fusion sequences can be placed in the appropriate expression vectors and used to express proteins in either bacteria or plants.

The nucleotide sequence of ESP1 contains three nucleotide differences and three corresponding amino acid differences for the ESP1 sequence disclosed in pending U.S. applications Ser. Nos. 08/888,950 and 08/888,949, both filed Jul. 7, 1997. Both the sequences disclosed in the present application and the sequences disclosed in the pending U.S. applications contain functional fumonisin esterase genes. For the purposes of the present invention, either the original ESP1 sequences or the ESP1 sequences disclosed may be used in combination with the APAO sequences or in fusion sequences. The nucleotide sequence of a BAA:ESP1:K:trAPAO construct for plant expression can be found in SEQ ID NO: 24 and the translation in SEQ ID NO: 25. The nucleotide sequence for a BAA:BEST1:K:trAPAO construct for plant expression can be found in SEQ ID NO: 26 and the translation in SEQ ID NO: 27. The nucleotide sequence of a GST:ESP1:K:trAPAO fusion for bacterial expression in a pGEX-4T-1 or similar vector can be found in SEQ ID NO: 28 and the translation in SEQ ID NO: 29. The nucleotide sequence for a GST:BEST1:K:trAPAO fusion for bacterial expression in a pGEX-4T-1 or similar vector can be seen in SEQ ID NO: 30 and the translation in SEQ ID NO: 31.

EXAMPLE 14 APAO Substrate Studies

The following assay was used to determine the substrate specificity of the APAO enzyme. Reaction mix: 436 μl of 200 mM Na-phosphate, pH8.0; 50 μl substrate (10 mM); 2 μl Amplex Red (1 mg in 200 μl DMSO); and 2 μl of Peroxidase (5000 U/ml). The APAO enzyme was recombinant enzyme produced as GST fusion in E. coli, purified over a glutathione affinity column and cleaved with thrombin to remove the GST. All components were mixed at room temperature. The initial rate was determined in a spectrophotometer at 572 nm over one minute by absorbance units/second (BLANK). Ten microliters of APAO at 70 ug/ml was added and mixed. The initial rate was again determined at 572 nm over one minute in absorbance units/second (SAMPLE). The rates were converted to absorbance units/minute. The BLANK value was subtracted from the SAMPLE value. The absorbance units were converted to μM H₂O₂ wherein 1 μM H₂O₂ equals 0.138 absorbance units at pH 8.0.

SUBSTRATES FOR APAO

RATE SUBSTRATE μM H₂O₂/min   1 mM Fumonisin B1 0.1429   1 mM AP1 0.8876 0.5 mg/mL Fumonisin B2 0.3058   1 mM Fumonisin B3 0.1449 0.5 mg/mL Fumonisin B4 0.1728   1 mM norepinephrine 0.0087   1 mM epinephrine 0.0071   1 mM dopamine 0.0040   1 mM spermine 0.0002

NOT SUBSTRATES FOR APAO (defined as compounds resulting in less than 1% conversion to hydrogen peroxide by APAO relative to AP1 under similar conditions of time, pH, temperature, and substrate concentration)

2-phenylethylamine, spermidine, EDTA-Na₂, tryptamine, putrescine, benzamidine, serotonin, cadaverine, Pefabloc SC, tyramine, 1,3-diaminopropane, leupeptin, histamine, hydroxylamine, aprotinin, deprenyl, Fumonisin C4, isoniazid, sphingosine, phenelzine, sphinganine, phytosphingosine, D-alanine, DL-alanine, L-arginine, L-asparagine, L-aspartic acid, D-aspartic acid, L-cysteine, L-glutamine, L-gltutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, DL-lysine, L-methionine, DL-methionine, L-phenylalanine, L-proline, L-threonine, L-tryptophan, L-tyrosine, L-valine.

EXAMPLE 15 Removal of Glycosylation Sites from APAO

Some cytosolic enzymes, when engineered for secretion by fusion with a heterologous signal peptide, lack function due to glycosylation at one or more potential glycosylation sites (amino acid consensus sequence N-X-S/T) that are not normally glycosylated in the native environment (Farrell L B, Beachy R N, Plant Mol Biol 15(6):821-5 (1990)). Since APAO lacks a recognizable signal sequence, it may be cytoplasmically localized in Exophiala spinifera, although secretion by some other method not involving a signal peptide cannot be ruled out. APAO contains two potential glycosylation sites, which can potentially be glycosylated, when APAO is secreted in a plant or other eukaryotic cell. These glycosylation sites can be eliminated without affecting protein function by site-directed mutagenesis using standard protocols (such as kits available from CLONTECH Laboratories, Inc. (Palo Alto, Calif.)).

SEQ ID NO: 33 shows the amino acid sequence of a GST:APAO in which two amino acids of APAO have been changed by site-directed mutagenesis to eliminate two potential glycosylation sites. The polynucleotide sequence of SEQ ID NO: 33 can be found in SEQ ID NO: 32. The first mutation changes asparagine at amino acid 201 of APAO to serine, and the second mutation changes serine at amino acid 206 of APAO to asparagine.Other mutations at either amino acid 200, 201, 202, 203, 204, 205, 206, or 207 of APAO, or a combination of these, can also be engineered to accomplish the removal of the glycosylation signal (Mellquist, J. L., Kasturi, L., Spitalnik, S. L., and Shakin-Eshelman, S. H., 1998. The amino acid following an Asn-X-Ser/Thr sequence is an important determinant of n-linked core glycosylation efficiency. Biochemistry 37:6833).

Other modifications to APAO can be made to improve its expression in a plant system, including site-directed mutagenesis to remove selected cysteine residues, which may be detrimental to proper folding when the protein is secreted into the endomembrane system for delivery to the apoplast. Cysteines are present at residues 64, 109, 167, 292, 351, 359, 387, 461, and 482, and may or may not be involved in disulfide crosslinking in mature, folded APAkO. Using standard methods of site-directed mutagenesis, one or more of these residues can be substituted with alanine or other suitable amino acid, resulting in a modified version of APAO that retains its activity and specificity but displays better activity and stability in an extracellular environment. It is possible that one or more cysteines is involved in covalent attachment of the FAD moiety to the APAO protein, and elimination of this cysteine would be expected to reduce or abolish activity.

All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

                   #             SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 33 <210> SEQ ID NO 1 <211> LENGTH: 372 <212> TYPE: DNA <213> ORGANISM: Exophiala spinifera. <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (346)...(346) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 1 gggccccggc gttctcgtag gctgcgcgga gttggtccca gacagacttt tg #tcgtacct     60 gcttggactg ttgggaccac ttccgtcccg ggtctccgac catgaaacag gt #aatggacc    120 attgtcgatc gacgtcgatg ctggtatctc tggcaaatga gatggggtca ca #gctcgatt    180 ggaggacgcc cgagaagcct tgttcgcgcc accacggctt gtcccatacg aa #gactatct    240 tgctatagta gcccaggata gaattttccg ccaatgcttg cttctcggcg gg #aagaggtg    300 gtgaaaatgt caaggtggga tacaaggttg tcggtaacga aaccancacc tt #tttgcttc    360 ggaacacggc gc               #                   #                   #      372 <210> SEQ ID NO 2 <211> LENGTH: 182 <212> TYPE: DNA <213> ORGANISM: Exophiala spinifera. <400> SEQUENCE: 2 gaattttccg ccaatgcttg cttctcggcg ggaagaggtg gtgaaaatgt ca #aggtggga     60 tacaaggttg tcggtaacga aaccaccacc tttttgcttc ggaacacggc gc #ccgaggcc    120 gatcgtactg tacagccgga tgccgactgc tcaatttcag cgacgggggt gt #tgaggtgc    180 ac                   #                   #                   #             182 <210> SEQ ID NO 3 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Designed oligonucleotide for # 3′ RACE, N21965 <400> SEQUENCE: 3 tggtttcgtt accgacaacc ttgtatccc          #                   #            29 <210> SEQ ID NO 4 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Designed oligonucleotide for # 5′ RACE, N21968 <400> SEQUENCE: 4 gagttggtcc cagacagact tttgtcgt          #                   #             28 <210> SEQ ID NO 5 <211> LENGTH: 1389 <212> TYPE: DNA <213> ORGANISM: Exophiala spinifera <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(1386) <400> SEQUENCE: 5 gac aac gtt gcg gac gtg gta gtg gtg ggc gc #t ggc ttg agc ggt ttg       48 Asp Asn Val Ala Asp Val Val Val Val Gly Al #a Gly Leu Ser Gly Leu  1               5   #                 10  #                 15 gag acg gca cgc aaa gtc cag gcc gcc ggt ct #g tcc tgc ctc gtt ctt       96 Glu Thr Ala Arg Lys Val Gln Ala Ala Gly Le #u Ser Cys Leu Val Leu              20      #             25      #             30 gag gcg atg gat cgt gta ggg gga aag act ct #g agc gta caa tcg ggt      144 Glu Ala Met Asp Arg Val Gly Gly Lys Thr Le #u Ser Val Gln Ser Gly          35          #         40          #         45 ccc ggc agg acg act atc aac gac ctc ggc gc #t gcg tgg atc aat gac      192 Pro Gly Arg Thr Thr Ile Asn Asp Leu Gly Al #a Ala Trp Ile Asn Asp      50              #     55              #     60 agc aac caa agc gaa gta tcc aga ttg ttt ga #a aga ttt cat ttg gag      240 Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Gl #u Arg Phe His Leu Glu  65                  # 70                  # 75                  # 80 ggc gag ctc cag agg acg act gga aat tca at #c cat caa gca caa gac      288 Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Il #e His Gln Ala Gln Asp                  85  #                 90  #                 95 ggt aca acc act aca gct cct tat ggt gac tc #c ttg ctg agc gag gag      336 Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Se #r Leu Leu Ser Glu Glu             100       #           105       #           110 gtt gca agt gca ctt gcg gaa ctc ctc ccc gt #a tgg tct cag ctg atc      384 Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Va #l Trp Ser Gln Leu Ile         115           #       120           #       125 gaa gag cat agc ctt caa gac ctc aag gcg ag #c cct cag gcg aag cgg      432 Glu Glu His Ser Leu Gln Asp Leu Lys Ala Se #r Pro Gln Ala Lys Arg     130               #   135               #   140 ctc gac agt gtg agc ttc gcg cac tac tgt ga #g aag gaa cta aac ttg      480 Leu Asp Ser Val Ser Phe Ala His Tyr Cys Gl #u Lys Glu Leu Asn Leu 145                 1 #50                 1 #55                 1 #60 cct gct gtt ctc ggc gta gca aac cag atc ac #a cgc gct ctg ctc ggt      528 Pro Ala Val Leu Gly Val Ala Asn Gln Ile Th #r Arg Ala Leu Leu Gly                 165   #               170   #               175 gtg gaa gcc cac gag atc agc atg ctt ttt ct #c acc gac tac atc aag      576 Val Glu Ala His Glu Ile Ser Met Leu Phe Le #u Thr Asp Tyr Ile Lys             180       #           185       #           190 agt gcc acc ggt ctc agt aat att ttc tcg ga #c aag aaa gac ggc ggg      624 Ser Ala Thr Gly Leu Ser Asn Ile Phe Ser As #p Lys Lys Asp Gly Gly         195           #       200           #       205 cag tat atg cga tgc aaa aca ggt atg cag tc #g att tgc cat gcc atg      672 Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Se #r Ile Cys His Ala Met     210               #   215               #   220 tca aag gaa ctt gtt cca ggc tca gtg cac ct #c aac acc ccc gtc gct      720 Ser Lys Glu Leu Val Pro Gly Ser Val His Le #u Asn Thr Pro Val Ala 225                 2 #30                 2 #35                 2 #40 gaa att gag cag tcg gca tcc ggc tgt aca gt #a cga tcg gcc tcg ggc      768 Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Va #l Arg Ser Ala Ser Gly                 245   #               250   #               255 gcc gtg ttc cga agc aaa aag gtg gtg gtt tc #g tta ccg aca acc ttg      816 Ala Val Phe Arg Ser Lys Lys Val Val Val Se #r Leu Pro Thr Thr Leu             260       #           265       #           270 tat ccc acc ttg aca ttt tca cca cct ctt cc #c gcc gag aag caa gca      864 Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pr #o Ala Glu Lys Gln Ala         275           #       280           #       285 ttg gcg gaa aat tct atc ctg ggc tac tat ag #c aag ata gtc ttc gta      912 Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Se #r Lys Ile Val Phe Val     290               #   295               #   300 tgg gac aag ccg tgg tgg cgc gaa caa ggc tt #c tcg ggc gtc ctc caa      960 Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Ph #e Ser Gly Val Leu Gln 305                 3 #10                 3 #15                 3 #20 tcg agc tgt gac ccc atc tca ttt gcc aga ga #t acc agc atc gac gtc     1008 Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg As #p Thr Ser Ile Asp Val                 325   #               330   #               335 gat cga caa tgg tcc att acc tgt ttc atg gt #c gga gac ccg gga cgg     1056 Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Va #l Gly Asp Pro Gly Arg             340       #           345       #           350 aag tgg tcc caa cag tcc aag cag gta cga ca #a aag tct gtc tgg gac     1104 Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gl #n Lys Ser Val Trp Asp         355           #       360           #       365 caa ctc cgc gca gcc tac gag aac gcc ggg gc #c caa gtc cca gag ccg     1152 Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly Al #a Gln Val Pro Glu Pro     370               #   375               #   380 gcc aac gtg ctc gaa atc gag tgg tcg aag ca #g cag tat ttc caa gga     1200 Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gl #n Gln Tyr Phe Gln Gly 385                 3 #90                 3 #95                 4 #00 gct ccg agc gcc gtc tat ggg ctg aac gat ct #c atc aca ctg ggt tcg     1248 Ala Pro Ser Ala Val Tyr Gly Leu Asn Asp Le #u Ile Thr Leu Gly Ser                 405   #               410   #               415 gcg ctc aga acg ccg ttc aag agt gtt cat tt #c gtt gga acg gag acg     1296 Ala Leu Arg Thr Pro Phe Lys Ser Val His Ph #e Val Gly Thr Glu Thr             420       #           425       #           430 tct tta gtt tgg aaa ggg tat atg gaa ggg gc #c ata cga tcg ggt caa     1344 Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Al #a Ile Arg Ser Gly Gln         435           #       440           #       445 cga ggt gct gca gaa gtt gtg gct agc ctg gt #g cca gca gca              #1386 Arg Gly Ala Ala Glu Val Val Ala Ser Leu Va #l Pro Ala Ala     450               #   455               #   460 tag                   #                   #                   #           1389 <210> SEQ ID NO 6 <211> LENGTH: 462 <212> TYPE: PRT <213> ORGANISM: Exophiala spinifera <400> SEQUENCE: 6 Asp Asn Val Ala Asp Val Val Val Val Gly Al #a Gly Leu Ser Gly Leu  1               5   #                10   #                15 Glu Thr Ala Arg Lys Val Gln Ala Ala Gly Le #u Ser Cys Leu Val Leu             20       #            25       #            30 Glu Ala Met Asp Arg Val Gly Gly Lys Thr Le #u Ser Val Gln Ser Gly         35           #        40           #        45 Pro Gly Arg Thr Thr Ile Asn Asp Leu Gly Al #a Ala Trp Ile Asn Asp     50               #    55               #    60 Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Gl #u Arg Phe His Leu Glu 65                   #70                   #75                   #80 Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Il #e His Gln Ala Gln Asp                 85   #                90   #                95 Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Se #r Leu Leu Ser Glu Glu             100       #           105       #           110 Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Va #l Trp Ser Gln Leu Ile         115           #       120           #       125 Glu Glu His Ser Leu Gln Asp Leu Lys Ala Se #r Pro Gln Ala Lys Arg     130               #   135               #   140 Leu Asp Ser Val Ser Phe Ala His Tyr Cys Gl #u Lys Glu Leu Asn Leu 145                 1 #50                 1 #55                 1 #60 Pro Ala Val Leu Gly Val Ala Asn Gln Ile Th #r Arg Ala Leu Leu Gly                 165   #               170   #               175 Val Glu Ala His Glu Ile Ser Met Leu Phe Le #u Thr Asp Tyr Ile Lys             180       #           185       #           190 Ser Ala Thr Gly Leu Ser Asn Ile Phe Ser As #p Lys Lys Asp Gly Gly         195           #       200           #       205 Gln Tyr Met Arg Cys Lys Thr Gly Met Gln Se #r Ile Cys His Ala Met     210               #   215               #   220 Ser Lys Glu Leu Val Pro Gly Ser Val His Le #u Asn Thr Pro Val Ala 225                 2 #30                 2 #35                 2 #40 Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Va #l Arg Ser Ala Ser Gly                 245   #               250   #               255 Ala Val Phe Arg Ser Lys Lys Val Val Val Se #r Leu Pro Thr Thr Leu             260       #           265       #           270 Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pr #o Ala Glu Lys Gln Ala         275           #       280           #       285 Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Se #r Lys Ile Val Phe Val     290               #   295               #   300 Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Ph #e Ser Gly Val Leu Gln 305                 3 #10                 3 #15                 3 #20 Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg As #p Thr Ser Ile Asp Val                 325   #               330   #               335 Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Va #l Gly Asp Pro Gly Arg             340       #           345       #           350 Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gl #n Lys Ser Val Trp Asp         355           #       360           #       365 Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly Al #a Gln Val Pro Glu Pro     370               #   375               #   380 Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gl #n Gln Tyr Phe Gln Gly 385                 3 #90                 3 #95                 4 #00 Ala Pro Ser Ala Val Tyr Gly Leu Asn Asp Le #u Ile Thr Leu Gly Ser                 405   #               410   #               415 Ala Leu Arg Thr Pro Phe Lys Ser Val His Ph #e Val Gly Thr Glu Thr             420       #           425       #           430 Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Al #a Ile Arg Ser Gly Gln         435           #       440           #       445 Arg Gly Ala Ala Glu Val Val Ala Ser Leu Va #l Pro Ala Ala     450               #   455               #   460 <210> SEQ ID NO 7 <211> LENGTH: 1442 <212> TYPE: DNA <213> ORGANISM: Exophiala spinifera <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(646) <221> NAME/KEY: intron <222> LOCATION: (647)...(699) <221> NAME/KEY: CDS <222> LOCATION: (700)...(1439) <400> SEQUENCE: 7 gac aac gtt gcg gac gtg gta gtg gtg ggc gc #t ggc ttg agc ggt ttg       48 Asp Asn Val Ala Asp Val Val Val Val Gly Al #a Gly Leu Ser Gly Leu  1               5   #                 10  #                 15 gag acg gca cgc aaa gtc cag gcc gcc ggt ct #g tcc tgc ctc gtt ctt       96 Glu Thr Ala Arg Lys Val Gln Ala Ala Gly Le #u Ser Cys Leu Val Leu              20      #             25      #             30 gag gcg atg gat cgt gta ggg gga aag act ct #g agc gta caa tcg ggt      144 Glu Ala Met Asp Arg Val Gly Gly Lys Thr Le #u Ser Val Gln Ser Gly          35          #         40          #         45 ccc ggc agg acg act atc aac gac ctc ggc gc #t gcg tgg atc aat gac      192 Pro Gly Arg Thr Thr Ile Asn Asp Leu Gly Al #a Ala Trp Ile Asn Asp      50              #     55              #     60 agc aac caa agc gaa gta tcc aga ttg ttt ga #a aga ttt cat ttg gag      240 Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Gl #u Arg Phe His Leu Glu  65                  # 70                  # 75                  # 80 ggc gag ctc cag agg acg act gga aat tca at #c cat caa gca caa gac      288 Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Il #e His Gln Ala Gln Asp                  85  #                 90  #                 95 ggt aca acc act aca gct cct tat ggt gac tc #c ttg ctg agc gag gag      336 Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Se #r Leu Leu Ser Glu Glu             100       #           105       #           110 gtt gca agt gca ctt gcg gaa ctc ctc ccc gt #a tgg tct cag ctg atc      384 Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Va #l Trp Ser Gln Leu Ile         115           #       120           #       125 gaa gag cat agc ctt caa gac ctc aag gcg ag #c cct cag gcg aag cgg      432 Glu Glu His Ser Leu Gln Asp Leu Lys Ala Se #r Pro Gln Ala Lys Arg     130               #   135               #   140 ctc gac agt gtg agc ttc gcg cac tac tgt ga #g aag gaa cta aac ttg      480 Leu Asp Ser Val Ser Phe Ala His Tyr Cys Gl #u Lys Glu Leu Asn Leu 145                 1 #50                 1 #55                 1 #60 cct gct gtt ctc ggc gta gca aac cag atc ac #a cgc gct ctg ctc ggt      528 Pro Ala Val Leu Gly Val Ala Asn Gln Ile Th #r Arg Ala Leu Leu Gly                 165   #               170   #               175 gtg gaa gcc cac gag atc agc atg ctt ttt ct #c acc gac tac atc aag      576 Val Glu Ala His Glu Ile Ser Met Leu Phe Le #u Thr Asp Tyr Ile Lys             180       #           185       #           190 agt gcc acc ggt ctc agt aat att ttc tcg ga #c aag aaa gac ggc ggg      624 Ser Ala Thr Gly Leu Ser Asn Ile Phe Ser As #p Lys Lys Asp Gly Gly         195           #       200           #       205 cag tat gtg cga tgc aaa aca g gtgcgtgtgg tgtc #gtctca ggtgggggac       676 Gln Tyr Val Arg Cys Lys Thr     210               #   215 tcgtttctca gtggtcattc cag gt atg cag tcg att tgc # cat gcc atg tca     728                    #       Gly Met Gln Ser Ile Cys H #is Ala Met Ser                    #                   #     220             #     225 aag gaa ctt gtt cca ggc tca gtg cac ctc aa #c acc ccc gtc gct gaa      776 Lys Glu Leu Val Pro Gly Ser Val His Leu As #n Thr Pro Val Ala Glu                 230   #               235   #               240 att gag cag tcg gca tcc ggc tgt aca gta cg #a tcg gcc tcg ggc gcc      824 Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Ar #g Ser Ala Ser Gly Ala             245       #           250       #           255 gtg ttc cga agc aaa aag gtg gtg gtt tcg tt #a ccg aca acc ttg tat      872 Val Phe Arg Ser Lys Lys Val Val Val Ser Le #u Pro Thr Thr Leu Tyr         260           #       265           #       270 ccc acc ttg aca ttt tca cca cct ctt ccc gc #c gag aag caa gca ttg      920 Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Al #a Glu Lys Gln Ala Leu     275               #   280               #   285 gcg gaa aat tct atc ctg ggc tac tat agc aa #g ata gtc ttc gta tgg      968 Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Ly #s Ile Val Phe Val Trp 290                 2 #95                 3 #00                 3 #05 gac aag ccg tgg tgg cgc gaa caa ggc ttc tc #g ggc gtc ctc caa tcg     1016 Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Se #r Gly Val Leu Gln Ser                 310   #               315   #               320 agc tgt gac ccc atc tca ttt gcc aga gat ac #c agc atc gac gtc gat     1064 Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Th #r Ser Ile Asp Val Asp             325       #           330       #           335 cga caa tgg tcc att acc tgt ttc atg gtc gg #a gac ccg gga cgg aag     1112 Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gl #y Asp Pro Gly Arg Lys         340           #       345           #       350 tgg tcc caa cag tcc aag cag gta cga caa aa #g tct gtc tgg gac caa     1160 Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Ly #s Ser Val Trp Asp Gln     355               #   360               #   365 ctc cgc gca gcc tac gag aac gcc ggg gcc ca #a gtc cca gag ccg gcc     1208 Leu Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gl #n Val Pro Glu Pro Ala 370                 3 #75                 3 #80                 3 #85 aac gtg ctc gaa atc gag tgg tcg aag cag ca #g tat ttc caa gga gct     1256 Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gl #n Tyr Phe Gln Gly Ala                 390   #               395   #               400 ccg agc gcc gtc tat ggg ctg aac gat ctc at #c aca ctg ggt tcg gcg     1304 Pro Ser Ala Val Tyr Gly Leu Asn Asp Leu Il #e Thr Leu Gly Ser Ala             405       #           410       #           415 ctc aga acg ccg ttc aag agt gtt cat ttc gt #t gga acg gag acg tct     1352 Leu Arg Thr Pro Phe Lys Ser Val His Phe Va #l Gly Thr Glu Thr Ser         420           #       425           #       430 tta gtt tgg aaa ggg tat atg gaa ggg gcc at #a cga tcg ggt caa cga     1400 Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Il #e Arg Ser Gly Gln Arg     435               #   440               #   445 ggt gct gca gaa gtt gtg gct agc ctg gtg cc #a gca gca tag              #1442 Gly Ala Ala Glu Val Val Ala Ser Leu Val Pr #o Ala Ala 450                 4 #55                 4 #60 <210> SEQ ID NO 8 <211> LENGTH: 462 <212> TYPE: PRT <213> ORGANISM: Exophiala spinifera <400> SEQUENCE: 8 Asp Asn Val Ala Asp Val Val Val Val Gly Al #a Gly Leu Ser Gly Leu  1               5   #                10   #                15 Glu Thr Ala Arg Lys Val Gln Ala Ala Gly Le #u Ser Cys Leu Val Leu             20       #            25       #            30 Glu Ala Met Asp Arg Val Gly Gly Lys Thr Le #u Ser Val Gln Ser Gly         35           #        40           #        45 Pro Gly Arg Thr Thr Ile Asn Asp Leu Gly Al #a Ala Trp Ile Asn Asp     50               #    55               #    60 Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Gl #u Arg Phe His Leu Glu 65                   #70                   #75                   #80 Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Il #e His Gln Ala Gln Asp                 85   #                90   #                95 Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Se #r Leu Leu Ser Glu Glu             100       #           105       #           110 Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Va #l Trp Ser Gln Leu Ile         115           #       120           #       125 Glu Glu His Ser Leu Gln Asp Leu Lys Ala Se #r Pro Gln Ala Lys Arg     130               #   135               #   140 Leu Asp Ser Val Ser Phe Ala His Tyr Cys Gl #u Lys Glu Leu Asn Leu 145                 1 #50                 1 #55                 1 #60 Pro Ala Val Leu Gly Val Ala Asn Gln Ile Th #r Arg Ala Leu Leu Gly                 165   #               170   #               175 Val Glu Ala His Glu Ile Ser Met Leu Phe Le #u Thr Asp Tyr Ile Lys             180       #           185       #           190 Ser Ala Thr Gly Leu Ser Asn Ile Phe Ser As #p Lys Lys Asp Gly Gly         195           #       200           #       205 Gln Tyr Val Arg Cys Lys Thr Gly Met Gln Se #r Ile Cys His Ala Met     210               #   215               #   220 Ser Lys Glu Leu Val Pro Gly Ser Val His Le #u Asn Thr Pro Val Ala 225                 2 #30                 2 #35                 2 #40 Glu Ile Glu Gln Ser Ala Ser Gly Cys Thr Va #l Arg Ser Ala Ser Gly                 245   #               250   #               255 Ala Val Phe Arg Ser Lys Lys Val Val Val Se #r Leu Pro Thr Thr Leu             260       #           265       #           270 Tyr Pro Thr Leu Thr Phe Ser Pro Pro Leu Pr #o Ala Glu Lys Gln Ala         275           #       280           #       285 Leu Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Se #r Lys Ile Val Phe Val     290               #   295               #   300 Trp Asp Lys Pro Trp Trp Arg Glu Gln Gly Ph #e Ser Gly Val Leu Gln 305                 3 #10                 3 #15                 3 #20 Ser Ser Cys Asp Pro Ile Ser Phe Ala Arg As #p Thr Ser Ile Asp Val                 325   #               330   #               335 Asp Arg Gln Trp Ser Ile Thr Cys Phe Met Va #l Gly Asp Pro Gly Arg             340       #           345       #           350 Lys Trp Ser Gln Gln Ser Lys Gln Val Arg Gl #n Lys Ser Val Trp Asp         355           #       360           #       365 Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gly Al #a Gln Val Pro Glu Pro     370               #   375               #   380 Ala Asn Val Leu Glu Ile Glu Trp Ser Lys Gl #n Gln Tyr Phe Gln Gly 385                 3 #90                 3 #95                 4 #00 Ala Pro Ser Ala Val Tyr Gly Leu Asn Asp Le #u Ile Thr Leu Gly Ser                 405   #               410   #               415 Ala Leu Arg Thr Pro Phe Lys Ser Val His Ph #e Val Gly Thr Glu Thr             420       #           425       #           430 Ser Leu Val Trp Lys Gly Tyr Met Glu Gly Al #a Ile Arg Ser Gly Gln         435           #       440           #       445 Arg Gly Ala Ala Glu Val Val Ala Ser Leu Va #l Pro Ala Ala     450               #   455               #   460 <210> SEQ ID NO 9 <211> LENGTH: 458 <212> TYPE: PRT <213> ORGANISM: Exophiala spinifera <400> SEQUENCE: 9 Asp Asn Val Ala Asp Val Val Val Val Gly Al #a Gly Leu Ser Gly Leu  1               5   #                10   #                15 Glu Thr Ala Arg Lys Val Gln Ala Ala Gly Le #u Ser Cys Leu Val Leu             20       #            25       #            30 Glu Ala Met Asp Arg Val Gly Gly Lys Thr Le #u Ser Val Gln Ser Gly         35           #        40           #        45 Pro Gly Arg Thr Thr Ile Asn Asp Leu Gly Al #a Ala Trp Ile Asn Asp     50               #    55               #    60 Ser Asn Gln Ser Glu Val Ser Arg Leu Phe Gl #u Arg Phe His Leu Glu 65                   #70                   #75                   #80 Gly Glu Leu Gln Arg Thr Thr Gly Asn Ser Il #e His Gln Ala Gln Asp                 85   #                90   #                95 Gly Thr Thr Thr Thr Ala Pro Tyr Gly Asp Se #r Leu Leu Ser Glu Glu             100       #           105       #           110 Val Ala Ser Ala Leu Ala Glu Leu Leu Pro Va #l Trp Ser Gln Leu Ile         115           #       120           #       125 Glu Glu His Ser Leu Gln Asp Leu Lys Ala Se #r Pro Gln Ala Lys Arg     130               #   135               #   140 Leu Asp Ser Val Ser Phe Ala His Tyr Cys Gl #u Lys Glu Leu Asn Leu 145                 1 #50                 1 #55                 1 #60 Pro Ala Val Leu Gly Val Ala Asn Gln Ile Th #r Arg Ala Leu Leu Gly                 165   #               170   #               175 Val Glu Ala His Glu Ile Ser Met Leu Phe Le #u Thr Asp Tyr Ile Lys             180       #           185       #           190 Ser Ala Thr Gly Leu Ser Asn Ile Phe Ser As #p Lys Lys Asp Gly Gly         195           #       200           #       205 Gln Tyr Val Arg Cys Lys Thr Gly Ala Cys Gl #y Val Val Ser Gly Gly     210               #   215               #   220 Gly Leu Val Ser Gln Trp Ser Phe Gln Val Cy #s Ser Arg Phe Ala Met 225                 2 #30                 2 #35                 2 #40 Pro Cys Gln Arg Asn Leu Phe Gln Ala Gln Cy #s Thr Ser Thr Pro Pro                 245   #               250   #               255 Ser Leu Lys Leu Ser Ser Arg His Pro Ala Va #l Gln Tyr Asp Arg Pro             260       #           265       #           270 Arg Ala Pro Cys Ser Glu Ala Lys Arg Trp Tr #p Phe Arg Tyr Arg Gln         275           #       280           #       285 Pro Cys Ile Pro Pro His Phe His His Leu Ph #e Pro Pro Arg Ser Lys     290               #   295               #   300 His Trp Arg Lys Ile Leu Ser Trp Ala Thr Il #e Ala Arg Ser Ser Tyr 305                 3 #10                 3 #15                 3 #20 Gly Thr Ser Arg Gly Gly Ala Asn Lys Ala Se #r Arg Ala Ser Ser Asn                 325   #               330   #               335 Arg Ala Val Thr Pro Ser His Leu Pro Glu Il #e Pro Ala Ser Thr Ser             340       #           345       #           350 Ile Asp Asn Gly Pro Leu Pro Val Ser Trp Se #r Glu Thr Arg Asp Gly         355           #       360           #       365 Ser Gly Pro Asn Ser Pro Ser Arg Tyr Asp Ly #s Ser Leu Ser Gly Thr     370               #   375               #   380 Asn Ser Ala Gln Pro Thr Arg Thr Pro Gly Pr #o Lys Ser Gln Ser Arg 385                 3 #90                 3 #95                 4 #00 Pro Thr Cys Ser Lys Ser Ser Gly Arg Ser Se #r Ser Ile Ser Lys Glu                 405   #               410   #               415 Leu Arg Ala Pro Ser Met Gly Thr Ile Ser Se #r His Trp Val Arg Arg             420       #           425       #           430 Ser Glu Arg Arg Ser Arg Val Phe Ile Ser Le #u Glu Arg Arg Arg Leu         435           #       440           #       445 Phe Gly Lys Gly Ile Trp Lys Gly Pro Tyr     450               #   455 <210> SEQ ID NO 10 <211> LENGTH: 1392 <212> TYPE: DNA <213> ORGANISM: Exophiala spinifera <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(1389) <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(3) <223> OTHER INFORMATION: Extra lysine in K:trAPAO <400> SEQUENCE: 10 aaa gac aac gtt gcg gac gtg gta gtg gtg gg #c gct ggc ttg agc ggt       48 Lys Asp Asn Val Ala Asp Val Val Val Val Gl #y Ala Gly Leu Ser Gly  1               5   #                 10  #                 15 ttg gag acg gca cgc aaa gtc cag gcc gcc gg #t ctg tcc tgc ctc gtt       96 Leu Glu Thr Ala Arg Lys Val Gln Ala Ala Gl #y Leu Ser Cys Leu Val              20      #             25      #             30 ctt gag gcg atg gat cgt gta ggg gga aag ac #t ctg agc gta caa tcg      144 Leu Glu Ala Met Asp Arg Val Gly Gly Lys Th #r Leu Ser Val Gln Ser          35          #         40          #         45 ggt ccc ggc agg acg act atc aac gac ctc gg #c gct gcg tgg atc aat      192 Gly Pro Gly Arg Thr Thr Ile Asn Asp Leu Gl #y Ala Ala Trp Ile Asn      50              #     55              #     60 gac agc aac caa agc gaa gta tcc aga ttg tt #t gaa aga ttt cat ttg      240 Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Ph #e Glu Arg Phe His Leu  65                  # 70                  # 75                  # 80 gag ggc gag ctc cag agg acg act gga aat tc #a atc cat caa gca caa      288 Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Se #r Ile His Gln Ala Gln                  85  #                 90  #                 95 gac ggt aca acc act aca gct cct tat ggt ga #c tcc ttg ctg agc gag      336 Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly As #p Ser Leu Leu Ser Glu             100       #           105       #           110 gag gtt gca agt gca ctt gcg gaa ctc ctc cc #c gta tgg tct cag ctg      384 Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pr #o Val Trp Ser Gln Leu         115           #       120           #       125 atc gaa gag cat agc ctt caa gac ctc aag gc #g agc cct cag gcg aag      432 Ile Glu Glu His Ser Leu Gln Asp Leu Lys Al #a Ser Pro Gln Ala Lys     130               #   135               #   140 cgg ctc gac agt gtg agc ttc gcg cac tac tg #t gag aag gaa cta aac      480 Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cy #s Glu Lys Glu Leu Asn 145                 1 #50                 1 #55                 1 #60 ttg cct gct gtt ctc ggc gta gca aac cag at #c aca cgc gct ctg ctc      528 Leu Pro Ala Val Leu Gly Val Ala Asn Gln Il #e Thr Arg Ala Leu Leu                 165   #               170   #               175 ggt gtg gaa gcc cac gag atc agc atg ctt tt #t ctc acc gac tac atc      576 Gly Val Glu Ala His Glu Ile Ser Met Leu Ph #e Leu Thr Asp Tyr Ile             180       #           185       #           190 aag agt gcc acc ggt ctc agt aat att ttc tc #g gac aag aaa gac ggc      624 Lys Ser Ala Thr Gly Leu Ser Asn Ile Phe Se #r Asp Lys Lys Asp Gly         195           #       200           #       205 ggg cag tat atg cga tgc aaa aca ggt atg ca #g tcg att tgc cat gcc      672 Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gl #n Ser Ile Cys His Ala     210               #   215               #   220 atg tca aag gaa ctt gtt cca ggc tca gtg ca #c ctc aac acc ccc gtc      720 Met Ser Lys Glu Leu Val Pro Gly Ser Val Hi #s Leu Asn Thr Pro Val 225                 2 #30                 2 #35                 2 #40 gct gaa att gag cag tcg gca tcc ggc tgt ac #a gta cga tcg gcc tcg      768 Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Th #r Val Arg Ser Ala Ser                 245   #               250   #               255 ggc gcc gtg ttc cga agc aaa aag gtg gtg gt #t tcg tta ccg aca acc      816 Gly Ala Val Phe Arg Ser Lys Lys Val Val Va #l Ser Leu Pro Thr Thr             260       #           265       #           270 ttg tat ccc acc ttg aca ttt tca cca cct ct #t ccc gcc gag aag caa      864 Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Le #u Pro Ala Glu Lys Gln         275           #       280           #       285 gca ttg gcg gaa aat tct atc ctg ggc tac ta #t agc aag ata gtc ttc      912 Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Ty #r Ser Lys Ile Val Phe     290               #   295               #   300 gta tgg gac aag ccg tgg tgg cgc gaa caa gg #c ttc tcg ggc gtc ctc      960 Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gl #y Phe Ser Gly Val Leu 305                 3 #10                 3 #15                 3 #20 caa tcg agc tgt gac ccc atc tca ttt gcc ag #a gat acc agc atc gac     1008 Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Ar #g Asp Thr Ser Ile Asp                 325   #               330   #               335 gtc gat cga caa tgg tcc att acc tgt ttc at #g gtc gga gac ccg gga     1056 Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Me #t Val Gly Asp Pro Gly             340       #           345       #           350 cgg aag tgg tcc caa cag tcc aag cag gta cg #a caa aag tct gtc tgg     1104 Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Ar #g Gln Lys Ser Val Trp         355           #       360           #       365 gac caa ctc cgc gca gcc tac gag aac gcc gg #g gcc caa gtc cca gag     1152 Asp Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gl #y Ala Gln Val Pro Glu     370               #   375               #   380 ccg gcc aac gtg ctc gaa atc gag tgg tcg aa #g cag cag tat ttc caa     1200 Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Ly #s Gln Gln Tyr Phe Gln 385                 3 #90                 3 #95                 4 #00 gga gct ccg agc gcc gtc tat ggg ctg aac ga #t ctc atc aca ctg ggt     1248 Gly Ala Pro Ser Ala Val Tyr Gly Leu Asn As #p Leu Ile Thr Leu Gly                 405   #               410   #               415 tcg gcg ctc aga acg ccg ttc aag agt gtt ca #t ttc gtt gga acg gag     1296 Ser Ala Leu Arg Thr Pro Phe Lys Ser Val Hi #s Phe Val Gly Thr Glu             420       #           425       #           430 acg tct tta gtt tgg aaa ggg tat atg gaa gg #g gcc ata cga tcg ggt     1344 Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gl #y Ala Ile Arg Ser Gly         435           #       440           #       445 caa cga ggt gct gca gaa gtt gtg gct agc ct #g gtg cca gca gca         1389 Gln Arg Gly Ala Ala Glu Val Val Ala Ser Le #u Val Pro Ala Ala     450               #   455               #   460 tag                   #                   #                   #           1392 <210> SEQ ID NO 11 <211> LENGTH: 463 <212> TYPE: PRT <213> ORGANISM: Exophiala spinifera <220> FEATURE: <223> OTHER INFORMATION: Extra lysine in the po #lypeptide sequence of       K:trAPAO, 463 aa. <400> SEQUENCE: 11 Lys Asp Asn Val Ala Asp Val Val Val Val Gl #y Ala Gly Leu Ser Gly  1               5   #                10   #                15 Leu Glu Thr Ala Arg Lys Val Gln Ala Ala Gl #y Leu Ser Cys Leu Val             20       #            25       #            30 Leu Glu Ala Met Asp Arg Val Gly Gly Lys Th #r Leu Ser Val Gln Ser         35           #        40           #        45 Gly Pro Gly Arg Thr Thr Ile Asn Asp Leu Gl #y Ala Ala Trp Ile Asn     50               #    55               #    60 Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Ph #e Glu Arg Phe His Leu 65                   #70                   #75                   #80 Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Se #r Ile His Gln Ala Gln                 85   #                90   #                95 Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly As #p Ser Leu Leu Ser Glu             100       #           105       #           110 Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pr #o Val Trp Ser Gln Leu         115           #       120           #       125 Ile Glu Glu His Ser Leu Gln Asp Leu Lys Al #a Ser Pro Gln Ala Lys     130               #   135               #   140 Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cy #s Glu Lys Glu Leu Asn 145                 1 #50                 1 #55                 1 #60 Leu Pro Ala Val Leu Gly Val Ala Asn Gln Il #e Thr Arg Ala Leu Leu                 165   #               170   #               175 Gly Val Glu Ala His Glu Ile Ser Met Leu Ph #e Leu Thr Asp Tyr Ile             180       #           185       #           190 Lys Ser Ala Thr Gly Leu Ser Asn Ile Phe Se #r Asp Lys Lys Asp Gly         195           #       200           #       205 Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gl #n Ser Ile Cys His Ala     210               #   215               #   220 Met Ser Lys Glu Leu Val Pro Gly Ser Val Hi #s Leu Asn Thr Pro Val 225                 2 #30                 2 #35                 2 #40 Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Th #r Val Arg Ser Ala Ser                 245   #               250   #               255 Gly Ala Val Phe Arg Ser Lys Lys Val Val Va #l Ser Leu Pro Thr Thr             260       #           265       #           270 Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Le #u Pro Ala Glu Lys Gln         275           #       280           #       285 Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Ty #r Ser Lys Ile Val Phe     290               #   295               #   300 Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gl #y Phe Ser Gly Val Leu 305                 3 #10                 3 #15                 3 #20 Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Ar #g Asp Thr Ser Ile Asp                 325   #               330   #               335 Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Me #t Val Gly Asp Pro Gly             340       #           345       #           350 Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Ar #g Gln Lys Ser Val Trp         355           #       360           #       365 Asp Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gl #y Ala Gln Val Pro Glu     370               #   375               #   380 Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Ly #s Gln Gln Tyr Phe Gln 385                 3 #90                 3 #95                 4 #00 Gly Ala Pro Ser Ala Val Tyr Gly Leu Asn As #p Leu Ile Thr Leu Gly                 405   #               410   #               415 Ser Ala Leu Arg Thr Pro Phe Lys Ser Val Hi #s Phe Val Gly Thr Glu             420       #           425       #           430 Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gl #y Ala Ile Arg Ser Gly         435           #       440           #       445 Gln Arg Gly Ala Ala Glu Val Val Ala Ser Le #u Val Pro Ala Ala     450               #   455               #   460 <210> SEQ ID NO 12 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer sequence designed  #for cloning DNA into       expression vectors, N23256 <400> SEQUENCE: 12 ggggaattca aagacaacgt tgcggacgtg gtag        #                   #        34 <210> SEQ ID NO 13 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer sequence designed  #for cloning DNA into       expression vectors, N23259 <400> SEQUENCE: 13 ggggcggccg cctatgctgc tggcaccagg ctag        #                   #        34 <210> SEQ ID NO 14 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Designed oligonucleotide for # 3′ RACE, N21965 <400> SEQUENCE: 14 tggtttcgtt accgacaacc ttgtatccc          #                   #            29 <210> SEQ ID NO 15 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Designed oligonucleotide for # 5′ race, N21968 <400> SEQUENCE: 15 gagttggtcc cagacagact tttgtcgt          #                   #             28 <210> SEQ ID NO 16 <211> LENGTH: 1673 <212> TYPE: DNA <213> ORGANISM: Exophiala spinifera <220> FEATURE: <221> NAME/KEY: sig_peptide <222> LOCATION: (1)...(267) <223> OTHER INFORMATION: yeast alpha mating factor # secretion signal. <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(1662) <400> SEQUENCE: 16 atg aga ttt cct tca att ttt act gct gtt tt #a ttc gca gca tcc tcc       48 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Le #u Phe Ala Ala Ser Ser                 -85   #               -80   #               -75 gca tta gct gct cca gtc aac act aca aca ga #a gat gaa acg gca caa       96 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Gl #u Asp Glu Thr Ala Gln             -70       #           -65       #           -60 att ccg gct gaa gct gtc atc ggt tac tca ga #t tta gaa ggg gat ttc      144 Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser As #p Leu Glu Gly Asp Phe         -55           #       -50           #       -45 gat gtt gct gtt ttg cca ttt tcc aac agc ac #a aat aac ggg tta ttg      192 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Th #r Asn Asn Gly Leu Leu     -40               #   -35               #   -30 ttt ata aat act act att gcc agc att gct gc #t aaa gaa gaa ggg gta      240 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Al #a Lys Glu Glu Gly Val -25                 - #20                 - #15                 - #10 tct ctc gag aaa aga gag gct gaa gct gaa tt #c aaa gac aac gtt gcg      288 Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Ph #e Lys Asp Asn Val Ala                  -5  #                1   #             5 gac gtg gta gtg gtg ggc gct ggc ttg agc gg #t ttg gag acg gca cgc      336 Asp Val Val Val Val Gly Ala Gly Leu Ser Gl #y Leu Glu Thr Ala Arg          10          #         15          #         20 aaa gtc cag gcc gcc ggt ctg tcc tgc ctc gt #t ctt gag gcg atg gat      384 Lys Val Gln Ala Ala Gly Leu Ser Cys Leu Va #l Leu Glu Ala Met Asp      25              #     30              #     35 cgt gta ggg gga aag act ctg agc gta caa tc #g ggt ccc ggc agg acg      432 Arg Val Gly Gly Lys Thr Leu Ser Val Gln Se #r Gly Pro Gly Arg Thr  40                  # 45                  # 50                  # 55 act atc aac gac ctc ggc gct gcg tgg atc aa #t gac agc aac caa agc      480 Thr Ile Asn Asp Leu Gly Ala Ala Trp Ile As #n Asp Ser Asn Gln Ser                  60  #                 65  #                 70 gaa gta tcc aga ttg ttt gaa aga ttt cat tt #g gag ggc gag ctc cag      528 Glu Val Ser Arg Leu Phe Glu Arg Phe His Le #u Glu Gly Glu Leu Gln              75      #             80      #             85 agg acg act gga aat tca atc cat caa gca ca #a gac ggt aca acc act      576 Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gl #n Asp Gly Thr Thr Thr          90          #         95          #        100 aca gct cct tat ggt gac tcc ttg ctg agc ga #g gag gtt gca agt gca      624 Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Gl #u Glu Val Ala Ser Ala     105               #   110               #   115 ctt gcg gaa ctc ctc ccc gta tgg tct cag ct #g atc gaa gag cat agc      672 Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Le #u Ile Glu Glu His Ser 120                 1 #25                 1 #30                 1 #35 ctt caa gac ctc aag gcg agc cct cag gcg aa #g cgg ctc gac agt gtg      720 Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Ly #s Arg Leu Asp Ser Val                 140   #               145   #               150 agc ttc gcg cac tac tgt gag aag gaa cta aa #c ttg cct gct gtt ctc      768 Ser Phe Ala His Tyr Cys Glu Lys Glu Leu As #n Leu Pro Ala Val Leu             155       #           160       #           165 ggc gta gca aac cag atc aca cgc gct ctg ct #c ggt gtg gaa gcc cac      816 Gly Val Ala Asn Gln Ile Thr Arg Ala Leu Le #u Gly Val Glu Ala His         170           #       175           #       180 gag atc agc atg ctt ttt ctc acc gac tac at #c aag agt gcc acc ggt      864 Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Il #e Lys Ser Ala Thr Gly     185               #   190               #   195 ctc agt aat att ttc tcg gac aag aaa gac gg #c ggg cag tat atg cga      912 Leu Ser Asn Ile Phe Ser Asp Lys Lys Asp Gl #y Gly Gln Tyr Met Arg 200                 2 #05                 2 #10                 2 #15 tgc aaa aca ggt atg cag tcg att tgc cat gc #c atg tca aag gaa ctt      960 Cys Lys Thr Gly Met Gln Ser Ile Cys His Al #a Met Ser Lys Glu Leu                 220   #               225   #               230 gtt cca ggc tca gtg cac ctc aac acc ccc gt #c gct gaa att gag cag     1008 Val Pro Gly Ser Val His Leu Asn Thr Pro Va #l Ala Glu Ile Glu Gln             235       #           240       #           245 tcg gca tcc ggc tgt aca gta cga tcg gcc tc #g ggc gcc gtg ttc cga     1056 Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Se #r Gly Ala Val Phe Arg         250           #       255           #       260 agc aaa aag gtg gtg gtt tcg tta ccg aca ac #c ttg tat ccc acc ttg     1104 Ser Lys Lys Val Val Val Ser Leu Pro Thr Th #r Leu Tyr Pro Thr Leu     265               #   270               #   275 aca ttt tca cca cct ctt ccc gcc gag aag ca #a gca ttg gcg gaa aat     1152 Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gl #n Ala Leu Ala Glu Asn 280                 2 #85                 2 #90                 2 #95 tct atc ctg ggc tac tat agc aag ata gtc tt #c gta tgg gac aag ccg     1200 Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Ph #e Val Trp Asp Lys Pro                 300   #               305   #               310 tgg tgg cgc gaa caa ggc ttc tcg ggc gtc ct #c caa tcg agc tgt gac     1248 Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Le #u Gln Ser Ser Cys Asp             315       #           320       #           325 ccc atc tca ttt gcc aga gat acc agc atc ga #c gtc gat cga caa tgg     1296 Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile As #p Val Asp Arg Gln Trp         330           #       335           #       340 tcc att acc tgt ttc atg gtc gga gac ccg gg #a cgg aag tgg tcc caa     1344 Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gl #y Arg Lys Trp Ser Gln     345               #   350               #   355 cag tcc aag cag gta cga caa aag tct gtc tg #g gac caa ctc cgc gca     1392 Gln Ser Lys Gln Val Arg Gln Lys Ser Val Tr #p Asp Gln Leu Arg Ala 360                 3 #65                 3 #70                 3 #75 gcc tac gag aac gcc ggg gcc caa gtc cca ga #g ccg gcc aac gtg ctc     1440 Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro Gl #u Pro Ala Asn Val Leu                 380   #               385   #               390 gaa atc gag tgg tcg aag cag cag tat ttc ca #a gga gct ccg agc gcc     1488 Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gl #n Gly Ala Pro Ser Ala             395       #           400       #           405 gtc tat ggg ctg aac gat ctc atc aca ctg gg #t tcg gcg ctc aga acg     1536 Val Tyr Gly Leu Asn Asp Leu Ile Thr Leu Gl #y Ser Ala Leu Arg Thr         410           #       415           #       420 ccg ttc aag agt gtt cat ttc gtt gga acg ga #g acg tct tta gtt tgg     1584 Pro Phe Lys Ser Val His Phe Val Gly Thr Gl #u Thr Ser Leu Val Trp     425               #   430               #   435 aaa ggg tat atg gaa ggg gcc ata cga tcg gg #t caa cga ggt gct gca     1632 Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gl #y Gln Arg Gly Ala Ala 440                 4 #45                 4 #50                 4 #55 gaa gtt gtg gct agc ctg gtg cca gca gca ta #ggcggccg c                # 1673 Glu Val Val Ala Ser Leu Val Pro Ala Ala                 460   #               465 <210> SEQ ID NO 17 <211> LENGTH: 554 <212> TYPE: PRT <213> ORGANISM: Exophiala spinifera <220> FEATURE: <221> NAME/KEY: SIGNAL <222> LOCATION: (1)...(89) <223> OTHER INFORMATION: yeast alpha mating factor # secretion signal. <400> SEQUENCE: 17 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Le #u Phe Ala Ala Ser Ser                 -85   #               -80   #               -75 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Gl #u Asp Glu Thr Ala Gln             -70       #           -65       #           -60 Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser As #p Leu Glu Gly Asp Phe         -55           #       -50           #       -45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Th #r Asn Asn Gly Leu Leu     -40               #   -35               #   -30 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Al #a Lys Glu Glu Gly Val -25                 - #20                 - #15                 - #10 Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Ph #e Lys Asp Asn Val Ala                 -5   #                 1  #              5 Asp Val Val Val Val Gly Ala Gly Leu Ser Gl #y Leu Glu Thr Ala Arg         10           #        15           #        20 Lys Val Gln Ala Ala Gly Leu Ser Cys Leu Va #l Leu Glu Ala Met Asp     25               #    30               #    35 Arg Val Gly Gly Lys Thr Leu Ser Val Gln Se #r Gly Pro Gly Arg Thr 40                   #45                   #50                   #55 Thr Ile Asn Asp Leu Gly Ala Ala Trp Ile As #n Asp Ser Asn Gln Ser                 60   #                65   #                70 Glu Val Ser Arg Leu Phe Glu Arg Phe His Le #u Glu Gly Glu Leu Gln             75       #            80       #            85 Arg Thr Thr Gly Asn Ser Ile His Gln Ala Gl #n Asp Gly Thr Thr Thr         90           #        95           #        100 Thr Ala Pro Tyr Gly Asp Ser Leu Leu Ser Gl #u Glu Val Ala Ser Ala     105               #   110               #   115 Leu Ala Glu Leu Leu Pro Val Trp Ser Gln Le #u Ile Glu Glu His Ser 120                 1 #25                 1 #30                 1 #35 Leu Gln Asp Leu Lys Ala Ser Pro Gln Ala Ly #s Arg Leu Asp Ser Val                 140   #               145   #               150 Ser Phe Ala His Tyr Cys Glu Lys Glu Leu As #n Leu Pro Ala Val Leu             155       #           160       #           165 Gly Val Ala Asn Gln Ile Thr Arg Ala Leu Le #u Gly Val Glu Ala His         170           #       175           #       180 Glu Ile Ser Met Leu Phe Leu Thr Asp Tyr Il #e Lys Ser Ala Thr Gly     185               #   190               #   195 Leu Ser Asn Ile Phe Ser Asp Lys Lys Asp Gl #y Gly Gln Tyr Met Arg 200                 2 #05                 2 #10                 2 #15 Cys Lys Thr Gly Met Gln Ser Ile Cys His Al #a Met Ser Lys Glu Leu                 220   #               225   #               230 Val Pro Gly Ser Val His Leu Asn Thr Pro Va #l Ala Glu Ile Glu Gln             235       #           240       #           245 Ser Ala Ser Gly Cys Thr Val Arg Ser Ala Se #r Gly Ala Val Phe Arg         250           #       255           #       260 Ser Lys Lys Val Val Val Ser Leu Pro Thr Th #r Leu Tyr Pro Thr Leu     265               #   270               #   275 Thr Phe Ser Pro Pro Leu Pro Ala Glu Lys Gl #n Ala Leu Ala Glu Asn 280                 2 #85                 2 #90                 2 #95 Ser Ile Leu Gly Tyr Tyr Ser Lys Ile Val Ph #e Val Trp Asp Lys Pro                 300   #               305   #               310 Trp Trp Arg Glu Gln Gly Phe Ser Gly Val Le #u Gln Ser Ser Cys Asp             315       #           320       #           325 Pro Ile Ser Phe Ala Arg Asp Thr Ser Ile As #p Val Asp Arg Gln Trp         330           #       335           #       340 Ser Ile Thr Cys Phe Met Val Gly Asp Pro Gl #y Arg Lys Trp Ser Gln     345               #   350               #   355 Gln Ser Lys Gln Val Arg Gln Lys Ser Val Tr #p Asp Gln Leu Arg Ala 360                 3 #65                 3 #70                 3 #75 Ala Tyr Glu Asn Ala Gly Ala Gln Val Pro Gl #u Pro Ala Asn Val Leu                 380   #               385   #               390 Glu Ile Glu Trp Ser Lys Gln Gln Tyr Phe Gl #n Gly Ala Pro Ser Ala             395       #           400       #           405 Val Tyr Gly Leu Asn Asp Leu Ile Thr Leu Gl #y Ser Ala Leu Arg Thr         410           #       415           #       420 Pro Phe Lys Ser Val His Phe Val Gly Thr Gl #u Thr Ser Leu Val Trp     425               #   430               #   435 Lys Gly Tyr Met Glu Gly Ala Ile Arg Ser Gl #y Gln Arg Gly Ala Ala 440                 4 #45                 4 #50                 4 #55 Glu Val Val Ala Ser Leu Val Pro Ala Ala                 460   #               465 <210> SEQ ID NO 18 <211> LENGTH: 2079 <212> TYPE: DNA <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(2076) <223> OTHER INFORMATION: GST:K:trAPAO 2079 nt. Tra #nslation starting at       nt 1 - 687, gst fusion +  #polylinker; 688-2076, K:trAPAO;       2077-2079, stop codon. For bacterial # expression. <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(687) <223> OTHER INFORMATION: gst fusion + polylinke #r <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (688)...(2076) <223> OTHER INFORMATION: K:trAPAO <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (688)...(690) <223> OTHER INFORMATION: Extra lysine <400> SEQUENCE: 18 atg tcc cct ata cta ggt tat tgg aaa att aa #g ggc ctt gtg caa ccc       48 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Ly #s Gly Leu Val Gln Pro  1               5   #                 10  #                 15 act cga ctt ctt ttg gaa tat ctt gaa gaa aa #a tat gaa gag cat ttg       96 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Ly #s Tyr Glu Glu His Leu              20      #             25      #             30 tat gag cgc gat gaa ggt gat aaa tgg cga aa #c aaa aag ttt gaa ttg      144 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg As #n Lys Lys Phe Glu Leu          35          #         40          #         45 ggt ttg gag ttt ccc aat ctt cct tat tat at #t gat ggt gat gtt aaa      192 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Il #e Asp Gly Asp Val Lys      50              #     55              #     60 tta aca cag tct atg gcc atc ata cgt tat at #a gct gac aag cac aac      240 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Il #e Ala Asp Lys His Asn  65                  # 70                  # 75                  # 80 atg ttg ggt ggt tgt cca aaa gag cgt gca ga #g att tca atg ctt gaa      288 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Gl #u Ile Ser Met Leu Glu                  85  #                 90  #                 95 gga gcg gtt ttg gat att aga tac ggt gtt tc #g aga att gca tat agt      336 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Se #r Arg Ile Ala Tyr Ser             100       #           105       #           110 aaa gac ttt gaa act ctc aaa gtt gat ttt ct #t agc aag cta cct gaa      384 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Le #u Ser Lys Leu Pro Glu         115           #       120           #       125 atg ctg aaa atg ttc gaa gat cgt tta tgt ca #t aaa aca tat tta aat      432 Met Leu Lys Met Phe Glu Asp Arg Leu Cys Hi #s Lys Thr Tyr Leu Asn     130               #   135               #   140 ggt gat cat gta acc cat cct gac ttc atg tt #g tat gac gct ctt gat      480 Gly Asp His Val Thr His Pro Asp Phe Met Le #u Tyr Asp Ala Leu Asp 145                 1 #50                 1 #55                 1 #60 gtt gtt tta tac atg gac cca atg tgc ctg ga #t gcg ttc cca aaa tta      528 Val Val Leu Tyr Met Asp Pro Met Cys Leu As #p Ala Phe Pro Lys Leu                 165   #               170   #               175 gtt tgt ttt aaa aaa cgt att gaa gct atc cc #a caa att gat aag tac      576 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pr #o Gln Ile Asp Lys Tyr             180       #           185       #           190 ttg aaa tcc agc aag tat ata gca tgg cct tt #g cag ggc tgg caa gcc      624 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Le #u Gln Gly Trp Gln Ala         195           #       200           #       205 acg ttt ggt ggt ggc gac cat cct cca aaa tc #g gat ctg gtt ccg cgt      672 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Se #r Asp Leu Val Pro Arg     210               #   215               #   220 gga tcc ccg gaa ttc aaa gac aac gtt gcg ga #c gtg gta gtg gtg ggc      720 Gly Ser Pro Glu Phe Lys Asp Asn Val Ala As #p Val Val Val Val Gly 225                 2 #30                 2 #35                 2 #40 gct ggc ttg agc ggt ttg gag acg gca cgc aa #a gtc cag gcc gcc ggt      768 Ala Gly Leu Ser Gly Leu Glu Thr Ala Arg Ly #s Val Gln Ala Ala Gly                 245   #               250   #               255 ctg tcc tgc ctc gtt ctt gag gcg atg gat cg #t gta ggg gga aag act      816 Leu Ser Cys Leu Val Leu Glu Ala Met Asp Ar #g Val Gly Gly Lys Thr             260       #           265       #           270 ctg agc gta caa tcg ggt ccc ggc agg acg ac #t atc aac gac ctc ggc      864 Leu Ser Val Gln Ser Gly Pro Gly Arg Thr Th #r Ile Asn Asp Leu Gly         275           #       280           #       285 gct gcg tgg atc aat gac agc aac caa agc ga #a gta tcc aga ttg ttt      912 Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Gl #u Val Ser Arg Leu Phe     290               #   295               #   300 gaa aga ttt cat ttg gag ggc gag ctc cag ag #g acg act gga aat tca      960 Glu Arg Phe His Leu Glu Gly Glu Leu Gln Ar #g Thr Thr Gly Asn Ser 305                 3 #10                 3 #15                 3 #20 atc cat caa gca caa gac ggt aca acc act ac #a gct cct tat ggt gac     1008 Ile His Gln Ala Gln Asp Gly Thr Thr Thr Th #r Ala Pro Tyr Gly Asp                 325   #               330   #               335 tcc ttg ctg agc gag gag gtt gca agt gca ct #t gcg gaa ctc ctc ccc     1056 Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Le #u Ala Glu Leu Leu Pro             340       #           345       #           350 gta tgg tct cag ctg atc gaa gag cat agc ct #t caa gac ctc aag gcg     1104 Val Trp Ser Gln Leu Ile Glu Glu His Ser Le #u Gln Asp Leu Lys Ala         355           #       360           #       365 agc cct cag gcg aag cgg ctc gac agt gtg ag #c ttc gcg cac tac tgt     1152 Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Se #r Phe Ala His Tyr Cys     370               #   375               #   380 gag aag gaa cta aac ttg cct gct gtt ctc gg #c gta gca aac cag atc     1200 Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gl #y Val Ala Asn Gln Ile 385                 3 #90                 3 #95                 4 #00 aca cgc gct ctg ctc ggt gtg gaa gcc cac ga #g atc agc atg ctt ttt     1248 Thr Arg Ala Leu Leu Gly Val Glu Ala His Gl #u Ile Ser Met Leu Phe                 405   #               410   #               415 ctc acc gac tac atc aag agt gcc acc ggt ct #c agt aat att ttc tcg     1296 Leu Thr Asp Tyr Ile Lys Ser Ala Thr Gly Le #u Ser Asn Ile Phe Ser             420       #           425       #           430 gac aag aaa gac ggc ggg cag tat atg cga tg #c aaa aca ggt atg cag     1344 Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cy #s Lys Thr Gly Met Gln         435           #       440           #       445 tcg att tgc cat gcc atg tca aag gaa ctt gt #t cca ggc tca gtg cac     1392 Ser Ile Cys His Ala Met Ser Lys Glu Leu Va #l Pro Gly Ser Val His     450               #   455               #   460 ctc aac acc ccc gtc gct gaa att gag cag tc #g gca tcc ggc tgt aca     1440 Leu Asn Thr Pro Val Ala Glu Ile Glu Gln Se #r Ala Ser Gly Cys Thr 465                 4 #70                 4 #75                 4 #80 gta cga tcg gcc tcg ggc gcc gtg ttc cga ag #c aaa aag gtg gtg gtt     1488 Val Arg Ser Ala Ser Gly Ala Val Phe Arg Se #r Lys Lys Val Val Val                 485   #               490   #               495 tcg tta ccg aca acc ttg tat ccc acc ttg ac #a ttt tca cca cct ctt     1536 Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Th #r Phe Ser Pro Pro Leu             500       #           505       #           510 ccc gcc gag aag caa gca ttg gcg gaa aat tc #t atc ctg ggc tac tat     1584 Pro Ala Glu Lys Gln Ala Leu Ala Glu Asn Se #r Ile Leu Gly Tyr Tyr         515           #       520           #       525 agc aag ata gtc ttc gta tgg gac aag ccg tg #g tgg cgc gaa caa ggc     1632 Ser Lys Ile Val Phe Val Trp Asp Lys Pro Tr #p Trp Arg Glu Gln Gly     530               #   535               #   540 ttc tcg ggc gtc ctc caa tcg agc tgt gac cc #c atc tca ttt gcc aga     1680 Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pr #o Ile Ser Phe Ala Arg 545                 5 #50                 5 #55                 5 #60 gat acc agc atc gac gtc gat cga caa tgg tc #c att acc tgt ttc atg     1728 Asp Thr Ser Ile Asp Val Asp Arg Gln Trp Se #r Ile Thr Cys Phe Met                 565   #               570   #               575 gtc gga gac ccg gga cgg aag tgg tcc caa ca #g tcc aag cag gta cga     1776 Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gl #n Ser Lys Gln Val Arg             580       #           585       #           590 caa aag tct gtc tgg gac caa ctc cgc gca gc #c tac gag aac gcc ggg     1824 Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Al #a Tyr Glu Asn Ala Gly         595           #       600           #       605 gcc caa gtc cca gag ccg gcc aac gtg ctc ga #a atc gag tgg tcg aag     1872 Ala Gln Val Pro Glu Pro Ala Asn Val Leu Gl #u Ile Glu Trp Ser Lys     610               #   615               #   620 cag cag tat ttc caa gga gct ccg agc gcc gt #c tat ggg ctg aac gat     1920 Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Va #l Tyr Gly Leu Asn Asp 625                 6 #30                 6 #35                 6 #40 ctc atc aca ctg ggt tcg gcg ctc aga acg cc #g ttc aag agt gtt cat     1968 Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pr #o Phe Lys Ser Val His                 645   #               650   #               655 ttc gtt gga acg gag acg tct tta gtt tgg aa #a ggg tat atg gaa ggg     2016 Phe Val Gly Thr Glu Thr Ser Leu Val Trp Ly #s Gly Tyr Met Glu Gly             660       #           665       #           670 gcc ata cga tcg ggt caa cga ggt gct gca ga #a gtt gtg gct agc ctg     2064 Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Gl #u Val Val Ala Ser Leu         675           #       680           #       685 gtg cca gca gca tag            #                   #                   #  2079 Val Pro Ala Ala     690 <210> SEQ ID NO 19 <211> LENGTH: 692 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: GST:K:trAPAO; GST + li #nker, aa 1-229; K:trAPAO,       aa 230-692 <400> SEQUENCE: 19 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Ly #s Gly Leu Val Gln Pro  1               5   #                10   #                15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Ly #s Tyr Glu Glu His Leu             20       #            25       #            30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg As #n Lys Lys Phe Glu Leu         35           #        40           #        45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Il #e Asp Gly Asp Val Lys     50               #    55               #    60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Il #e Ala Asp Lys His Asn 65                   #70                   #75                   #80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Gl #u Ile Ser Met Leu Glu                 85   #                90   #                95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Se #r Arg Ile Ala Tyr Ser             100       #           105       #           110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Le #u Ser Lys Leu Pro Glu         115           #       120           #       125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys Hi #s Lys Thr Tyr Leu Asn     130               #   135               #   140 Gly Asp His Val Thr His Pro Asp Phe Met Le #u Tyr Asp Ala Leu Asp 145                 1 #50                 1 #55                 1 #60 Val Val Leu Tyr Met Asp Pro Met Cys Leu As #p Ala Phe Pro Lys Leu                 165   #               170   #               175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pr #o Gln Ile Asp Lys Tyr             180       #           185       #           190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Le #u Gln Gly Trp Gln Ala         195           #       200           #       205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Se #r Asp Leu Val Pro Arg     210               #   215               #   220 Gly Ser Pro Glu Phe Lys Asp Asn Val Ala As #p Val Val Val Val Gly 225                 2 #30                 2 #35                 2 #40 Ala Gly Leu Ser Gly Leu Glu Thr Ala Arg Ly #s Val Gln Ala Ala Gly                 245   #               250   #               255 Leu Ser Cys Leu Val Leu Glu Ala Met Asp Ar #g Val Gly Gly Lys Thr             260       #           265       #           270 Leu Ser Val Gln Ser Gly Pro Gly Arg Thr Th #r Ile Asn Asp Leu Gly         275           #       280           #       285 Ala Ala Trp Ile Asn Asp Ser Asn Gln Ser Gl #u Val Ser Arg Leu Phe     290               #   295               #   300 Glu Arg Phe His Leu Glu Gly Glu Leu Gln Ar #g Thr Thr Gly Asn Ser 305                 3 #10                 3 #15                 3 #20 Ile His Gln Ala Gln Asp Gly Thr Thr Thr Th #r Ala Pro Tyr Gly Asp                 325   #               330   #               335 Ser Leu Leu Ser Glu Glu Val Ala Ser Ala Le #u Ala Glu Leu Leu Pro             340       #           345       #           350 Val Trp Ser Gln Leu Ile Glu Glu His Ser Le #u Gln Asp Leu Lys Ala         355           #       360           #       365 Ser Pro Gln Ala Lys Arg Leu Asp Ser Val Se #r Phe Ala His Tyr Cys     370               #   375               #   380 Glu Lys Glu Leu Asn Leu Pro Ala Val Leu Gl #y Val Ala Asn Gln Ile 385                 3 #90                 3 #95                 4 #00 Thr Arg Ala Leu Leu Gly Val Glu Ala His Gl #u Ile Ser Met Leu Phe                 405   #               410   #               415 Leu Thr Asp Tyr Ile Lys Ser Ala Thr Gly Le #u Ser Asn Ile Phe Ser             420       #           425       #           430 Asp Lys Lys Asp Gly Gly Gln Tyr Met Arg Cy #s Lys Thr Gly Met Gln         435           #       440           #       445 Ser Ile Cys His Ala Met Ser Lys Glu Leu Va #l Pro Gly Ser Val His     450               #   455               #   460 Leu Asn Thr Pro Val Ala Glu Ile Glu Gln Se #r Ala Ser Gly Cys Thr 465                 4 #70                 4 #75                 4 #80 Val Arg Ser Ala Ser Gly Ala Val Phe Arg Se #r Lys Lys Val Val Val                 485   #               490   #               495 Ser Leu Pro Thr Thr Leu Tyr Pro Thr Leu Th #r Phe Ser Pro Pro Leu             500       #           505       #           510 Pro Ala Glu Lys Gln Ala Leu Ala Glu Asn Se #r Ile Leu Gly Tyr Tyr         515           #       520           #       525 Ser Lys Ile Val Phe Val Trp Asp Lys Pro Tr #p Trp Arg Glu Gln Gly     530               #   535               #   540 Phe Ser Gly Val Leu Gln Ser Ser Cys Asp Pr #o Ile Ser Phe Ala Arg 545                 5 #50                 5 #55                 5 #60 Asp Thr Ser Ile Asp Val Asp Arg Gln Trp Se #r Ile Thr Cys Phe Met                 565   #               570   #               575 Val Gly Asp Pro Gly Arg Lys Trp Ser Gln Gl #n Ser Lys Gln Val Arg             580       #           585       #           590 Gln Lys Ser Val Trp Asp Gln Leu Arg Ala Al #a Tyr Glu Asn Ala Gly         595           #       600           #       605 Ala Gln Val Pro Glu Pro Ala Asn Val Leu Gl #u Ile Glu Trp Ser Lys     610               #   615               #   620 Gln Gln Tyr Phe Gln Gly Ala Pro Ser Ala Va #l Tyr Gly Leu Asn Asp 625                 6 #30                 6 #35                 6 #40 Leu Ile Thr Leu Gly Ser Ala Leu Arg Thr Pr #o Phe Lys Ser Val His                 645   #               650   #               655 Phe Val Gly Thr Glu Thr Ser Leu Val Trp Ly #s Gly Tyr Met Glu Gly             660       #           665       #           670 Ala Ile Arg Ser Gly Gln Arg Gly Ala Ala Gl #u Val Val Ala Ser Leu         675           #       680           #       685 Val Pro Ala Ala     690 <210> SEQ ID NO 20 <211> LENGTH: 1464 <212> TYPE: DNA <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: sig_peptide <222> LOCATION: (1)...(72) <223> OTHER INFORMATION: Barley Alpha Amylase sign #al sequence. <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (73)...(1464) <223> OTHER INFORMATION: K:trAPAOcDNA <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(1461) <223> OTHER INFORMATION: Nucleotide sequence of K: #trAPAO translational       fusion with barley alpha amylase  #signal sequence, for expression       and secretion of the mature trAPA #O in maize. Nucleotides 1-72,       barley alpha amylase signal sequence #, nucleotides 73-75, added       lysine residue; nucleotides 76 -1464 # , trAPAO cDNA. <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (73)...(75) <223> OTHER INFORMATION: Added lysine residue <400> SEQUENCE: 20 atg gcc aac aag cac ctg agc ctc tcc ctc tt #c ctc gtg ctc ctc ggc       48 Met Ala Asn Lys His Leu Ser Leu Ser Leu Ph #e Leu Val Leu Leu Gly                 -20   #               -15   #               -10 ctc tcc gcc tcc ctc gcc agc ggc aaa gac aa #c gtt gcg gac gtg gta       96 Leu Ser Ala Ser Leu Ala Ser Gly Lys Asp As #n Val Ala Asp Val Val              -5      #            1       #         5 gtg gtg ggc gct ggc ttg agc ggt ttg gag ac #g gca cgc aaa gtc cag      144 Val Val Gly Ala Gly Leu Ser Gly Leu Glu Th #r Ala Arg Lys Val Gln      10              #     15              #     20 gcc gcc ggt ctg tcc tgc ctc gtt ctt gag gc #g atg gat cgt gta ggg      192 Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Al #a Met Asp Arg Val Gly  25                  # 30                  # 35                  # 40 gga aag act ctg agc gta caa tcg ggt ccc gg #c agg acg act atc aac      240 Gly Lys Thr Leu Ser Val Gln Ser Gly Pro Gl #y Arg Thr Thr Ile Asn                  45  #                 50  #                 55 gac ctc ggc gct gcg tgg atc aat gac agc aa #c caa agc gaa gta tcc      288 Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser As #n Gln Ser Glu Val Ser              60      #             65      #             70 aga ttg ttt gaa aga ttt cat ttg gag ggc ga #g ctc cag agg acg act      336 Arg Leu Phe Glu Arg Phe His Leu Glu Gly Gl #u Leu Gln Arg Thr Thr          75          #         80          #         85 gga aat tca atc cat caa gca caa gac ggt ac #a acc act aca gct cct      384 Gly Asn Ser Ile His Gln Ala Gln Asp Gly Th #r Thr Thr Thr Ala Pro      90              #     95              #    100 tat ggt gac tcc ttg ctg agc gag gag gtt gc #a agt gca ctt gcg gaa      432 Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Al #a Ser Ala Leu Ala Glu 105                 1 #10                 1 #15                 1 #20 ctc ctc ccc gta tgg tct cag ctg atc gaa ga #g cat agc ctt caa gac      480 Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Gl #u His Ser Leu Gln Asp                 125   #               130   #               135 ctc aag gcg agc cct cag gcg aag cgg ctc ga #c agt gtg agc ttc gcg      528 Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu As #p Ser Val Ser Phe Ala             140       #           145       #           150 cac tac tgt gag aag gaa cta aac ttg cct gc #t gtt ctc ggc gta gca      576 His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Al #a Val Leu Gly Val Ala         155           #       160           #       165 aac cag atc aca cgc gct ctg ctc ggt gtg ga #a gcc cac gag atc agc      624 Asn Gln Ile Thr Arg Ala Leu Leu Gly Val Gl #u Ala His Glu Ile Ser     170               #   175               #   180 atg ctt ttt ctc acc gac tac atc aag agt gc #c acc ggt ctc agt aat      672 Met Leu Phe Leu Thr Asp Tyr Ile Lys Ser Al #a Thr Gly Leu Ser Asn 185                 1 #90                 1 #95                 2 #00 att ttc tcg gac aag aaa gac ggc ggg cag ta #t atg cga tgc aaa aca      720 Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Ty #r Met Arg Cys Lys Thr                 205   #               210   #               215 ggt atg cag tcg att tgc cat gcc atg tca aa #g gaa ctt gtt cca ggc      768 Gly Met Gln Ser Ile Cys His Ala Met Ser Ly #s Glu Leu Val Pro Gly             220       #           225       #           230 tca gtg cac ctc aac acc ccc gtc gct gaa at #t gag cag tcg gca tcc      816 Ser Val His Leu Asn Thr Pro Val Ala Glu Il #e Glu Gln Ser Ala Ser         235           #       240           #       245 ggc tgt aca gta cga tcg gcc tcg ggc gcc gt #g ttc cga agc aaa aag      864 Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Va #l Phe Arg Ser Lys Lys     250               #   255               #   260 gtg gtg gtt tcg tta ccg aca acc ttg tat cc #c acc ttg aca ttt tca      912 Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pr #o Thr Leu Thr Phe Ser 265                 2 #70                 2 #75                 2 #80 cca cct ctt ccc gcc gag aag caa gca ttg gc #g gaa aat tct atc ctg      960 Pro Pro Leu Pro Ala Glu Lys Gln Ala Leu Al #a Glu Asn Ser Ile Leu                 285   #               290   #               295 ggc tac tat agc aag ata gtc ttc gta tgg ga #c aag ccg tgg tgg cgc     1008 Gly Tyr Tyr Ser Lys Ile Val Phe Val Trp As #p Lys Pro Trp Trp Arg             300       #           305       #           310 gaa caa ggc ttc tcg ggc gtc ctc caa tcg ag #c tgt gac ccc atc tca     1056 Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Se #r Cys Asp Pro Ile Ser         315           #       320           #       325 ttt gcc aga gat acc agc atc gac gtc gat cg #a caa tgg tcc att acc     1104 Phe Ala Arg Asp Thr Ser Ile Asp Val Asp Ar #g Gln Trp Ser Ile Thr     330               #   335               #   340 tgt ttc atg gtc gga gac ccg gga cgg aag tg #g tcc caa cag tcc aag     1152 Cys Phe Met Val Gly Asp Pro Gly Arg Lys Tr #p Ser Gln Gln Ser Lys 345                 3 #50                 3 #55                 3 #60 cag gta cga caa aag tct gtc tgg gac caa ct #c cgc gca gcc tac gag     1200 Gln Val Arg Gln Lys Ser Val Trp Asp Gln Le #u Arg Ala Ala Tyr Glu                 365   #               370   #               375 aac gcc ggg gcc caa gtc cca gag ccg gcc aa #c gtg ctc gaa atc gag     1248 Asn Ala Gly Ala Gln Val Pro Glu Pro Ala As #n Val Leu Glu Ile Glu             380       #           385       #           390 tgg tcg aag cag cag tat ttc caa gga gct cc #g agc gcc gtc tat ggg     1296 Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pr #o Ser Ala Val Tyr Gly         395           #       400           #       405 ctg aac gat ctc atc aca ctg ggt tcg gcg ct #c aga acg ccg ttc aag     1344 Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Le #u Arg Thr Pro Phe Lys     410               #   415               #   420 agt gtt cat ttc gtt gga acg gag acg tct tt #a gtt tgg aaa ggg tat     1392 Ser Val His Phe Val Gly Thr Glu Thr Ser Le #u Val Trp Lys Gly Tyr 425                 4 #30                 4 #35                 4 #40 atg gaa ggg gcc ata cga tcg ggt caa cga gg #t gct gca gaa gtt gtg     1440 Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gl #y Ala Ala Glu Val Val                 445   #               450   #               455 gct agc ctg gtg cca gca gca tag      #                   #              1464 Ala Ser Leu Val Pro Ala Ala             460 <210> SEQ ID NO 21 <211> LENGTH: 487 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: SIGNAL <222> LOCATION: (1)...(24) <223> OTHER INFORMATION: K:trAPAO translational fusio #n with barley alpha       amylase signal sequence, for express #ion and secretion of the       mature trAPAO in maize. <400> SEQUENCE: 21 Met Ala Asn Lys His Leu Ser Leu Ser Leu Ph #e Leu Val Leu Leu Gly                 -20   #               -15   #               -10 Leu Ser Ala Ser Leu Ala Ser Gly Lys Asp As #n Val Ala Asp Val Val             -5       #             1      #          5 Val Val Gly Ala Gly Leu Ser Gly Leu Glu Th #r Ala Arg Lys Val Gln     10               #    15               #    20 Ala Ala Gly Leu Ser Cys Leu Val Leu Glu Al #a Met Asp Arg Val Gly 25                   #30                   #35                   #40 Gly Lys Thr Leu Ser Val Gln Ser Gly Pro Gl #y Arg Thr Thr Ile Asn                 45   #                50   #                55 Asp Leu Gly Ala Ala Trp Ile Asn Asp Ser As #n Gln Ser Glu Val Ser             60       #            65       #            70 Arg Leu Phe Glu Arg Phe His Leu Glu Gly Gl #u Leu Gln Arg Thr Thr         75           #        80           #        85 Gly Asn Ser Ile His Gln Ala Gln Asp Gly Th #r Thr Thr Thr Ala Pro     90               #    95               #    100 Tyr Gly Asp Ser Leu Leu Ser Glu Glu Val Al #a Ser Ala Leu Ala Glu 105                 1 #10                 1 #15                 1 #20 Leu Leu Pro Val Trp Ser Gln Leu Ile Glu Gl #u His Ser Leu Gln Asp                 125   #               130   #               135 Leu Lys Ala Ser Pro Gln Ala Lys Arg Leu As #p Ser Val Ser Phe Ala             140       #           145       #           150 His Tyr Cys Glu Lys Glu Leu Asn Leu Pro Al #a Val Leu Gly Val Ala         155           #       160           #       165 Asn Gln Ile Thr Arg Ala Leu Leu Gly Val Gl #u Ala His Glu Ile Ser     170               #   175               #   180 Met Leu Phe Leu Thr Asp Tyr Ile Lys Ser Al #a Thr Gly Leu Ser Asn 185                 1 #90                 1 #95                 2 #00 Ile Phe Ser Asp Lys Lys Asp Gly Gly Gln Ty #r Met Arg Cys Lys Thr                 205   #               210   #               215 Gly Met Gln Ser Ile Cys His Ala Met Ser Ly #s Glu Leu Val Pro Gly             220       #           225       #           230 Ser Val His Leu Asn Thr Pro Val Ala Glu Il #e Glu Gln Ser Ala Ser         235           #       240           #       245 Gly Cys Thr Val Arg Ser Ala Ser Gly Ala Va #l Phe Arg Ser Lys Lys     250               #   255               #   260 Val Val Val Ser Leu Pro Thr Thr Leu Tyr Pr #o Thr Leu Thr Phe Ser 265                 2 #70                 2 #75                 2 #80 Pro Pro Leu Pro Ala Glu Lys Gln Ala Leu Al #a Glu Asn Ser Ile Leu                 285   #               290   #               295 Gly Tyr Tyr Ser Lys Ile Val Phe Val Trp As #p Lys Pro Trp Trp Arg             300       #           305       #           310 Glu Gln Gly Phe Ser Gly Val Leu Gln Ser Se #r Cys Asp Pro Ile Ser         315           #       320           #       325 Phe Ala Arg Asp Thr Ser Ile Asp Val Asp Ar #g Gln Trp Ser Ile Thr     330               #   335               #   340 Cys Phe Met Val Gly Asp Pro Gly Arg Lys Tr #p Ser Gln Gln Ser Lys 345                 3 #50                 3 #55                 3 #60 Gln Val Arg Gln Lys Ser Val Trp Asp Gln Le #u Arg Ala Ala Tyr Glu                 365   #               370   #               375 Asn Ala Gly Ala Gln Val Pro Glu Pro Ala As #n Val Leu Glu Ile Glu             380       #           385       #           390 Trp Ser Lys Gln Gln Tyr Phe Gln Gly Ala Pr #o Ser Ala Val Tyr Gly         395           #       400           #       405 Leu Asn Asp Leu Ile Thr Leu Gly Ser Ala Le #u Arg Thr Pro Phe Lys     410               #   415               #   420 Ser Val His Phe Val Gly Thr Glu Thr Ser Le #u Val Trp Lys Gly Tyr 425                 4 #30                 4 #35                 4 #40 Met Glu Gly Ala Ile Arg Ser Gly Gln Arg Gl #y Ala Ala Glu Val Val                 445   #               450   #               455 Ala Ser Leu Val Pro Ala Ala             460 <210> SEQ ID NO 22 <211> LENGTH: 1803 <212> TYPE: DNA <213> ORGANISM: Exophiala spinifera <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(1800) <400> SEQUENCE: 22 atg gca ctt gca ccg agc tac atc aat ccc cc #a aac gtc gcc tcc cca       48 Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pr #o Asn Val Ala Ser Pro  1               5   #                 10  #                 15 gca ggg tat tct cac gtc ggc gta ggc cca ga #c gga ggg agg tat gtg       96 Ala Gly Tyr Ser His Val Gly Val Gly Pro As #p Gly Gly Arg Tyr Val              20      #             25      #             30 aca ata gct gga cag att gga caa gac gct tc #g ggc gtg aca gac cct      144 Thr Ile Ala Gly Gln Ile Gly Gln Asp Ala Se #r Gly Val Thr Asp Pro          35          #         40          #         45 gcc tac gag aaa cag gtt gcc caa gca ttc gc #c aat ctg cga gct tgc      192 Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Al #a Asn Leu Arg Ala Cys      50              #     55              #     60 ctt gct gca gtt gga gcc act tca aac gac gt #c acc aag ctc aat tac      240 Leu Ala Ala Val Gly Ala Thr Ser Asn Asp Va #l Thr Lys Leu Asn Tyr  65                  # 70                  # 75                  # 80 tac atc gtc gac tac gcc ccg agc aaa ctc ac #c gca att gga gat ggg      288 Tyr Ile Val Asp Tyr Ala Pro Ser Lys Leu Th #r Ala Ile Gly Asp Gly                  85  #                 90  #                 95 ctg aag gct acc ttt gcc ctt gac agg ctc cc #t cct tgc acg ctg gtg      336 Leu Lys Ala Thr Phe Ala Leu Asp Arg Leu Pr #o Pro Cys Thr Leu Val             100       #           105       #           110 cca gtg tcg gcc ttg tct tca cct gaa tac ct #c ttt gag gtt gat gcc      384 Pro Val Ser Ala Leu Ser Ser Pro Glu Tyr Le #u Phe Glu Val Asp Ala         115           #       120           #       125 acg gcg ctg gtg ccg gga cac acg acc cca ga #c aac gtt gcg gac gtg      432 Thr Ala Leu Val Pro Gly His Thr Thr Pro As #p Asn Val Ala Asp Val     130               #   135               #   140 gta gtg gtg ggc gct ggc ttg agc ggt ttg ga #g acg gca cgc aaa gtc      480 Val Val Val Gly Ala Gly Leu Ser Gly Leu Gl #u Thr Ala Arg Lys Val 145                 1 #50                 1 #55                 1 #60 cag gcc gcc ggt ctg tcc tgc ctc gtt ctt ga #g gcg atg gat cgt gta      528 Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Gl #u Ala Met Asp Arg Val                 165   #               170   #               175 ggg gga aag act ctg agc gta caa tcg ggt cc #c ggc agg acg act atc      576 Gly Gly Lys Thr Leu Ser Val Gln Ser Gly Pr #o Gly Arg Thr Thr Ile             180       #           185       #           190 aac gac ctc ggc gct gcg tgg atc aat gac ag #c aac caa agc gaa gta      624 Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Se #r Asn Gln Ser Glu Val         195           #       200           #       205 tcc aga ttg ttt gaa aga ttt cat ttg gag gg #c gag ctc cag agg acg      672 Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gl #y Glu Leu Gln Arg Thr     210               #   215               #   220 act gga aat tca atc cat caa gca caa gac gg #t aca acc act aca gct      720 Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gl #y Thr Thr Thr Thr Ala 225                 2 #30                 2 #35                 2 #40 cct tat ggt gac tcc ttg ctg agc gag gag gt #t gca agt gca ctt gcg      768 Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Va #l Ala Ser Ala Leu Ala                 245   #               250   #               255 gaa ctc ctc ccc gta tgg tct cag ctg atc ga #a gag cat agc ctt caa      816 Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Gl #u Glu His Ser Leu Gln             260       #           265       #           270 gac ctc aag gcg agc cct cag gcg aag cgg ct #c gac agt gtg agc ttc      864 Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Le #u Asp Ser Val Ser Phe         275           #       280           #       285 gcg cac tac tgt gag aag gaa cta aac ttg cc #t gct gtt ctc ggc gta      912 Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pr #o Ala Val Leu Gly Val     290               #   295               #   300 gca aac cag atc aca cgc gct ctg ctc ggt gt #g gaa gcc cac gag atc      960 Ala Asn Gln Ile Thr Arg Ala Leu Leu Gly Va #l Glu Ala His Glu Ile 305                 3 #10                 3 #15                 3 #20 agc atg ctt ttt ctc acc gac tac atc aag ag #t gcc acc ggt ctc agt     1008 Ser Met Leu Phe Leu Thr Asp Tyr Ile Lys Se #r Ala Thr Gly Leu Ser                 325   #               330   #               335 aat att ttc tcg gac aag aaa gac ggc ggg ca #g tat atg cga tgc aaa     1056 Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gl #n Tyr Met Arg Cys Lys             340       #           345       #           350 aca ggt atg cag tcg att tgc cat gcc atg tc #a aag gaa ctt gtt cca     1104 Thr Gly Met Gln Ser Ile Cys His Ala Met Se #r Lys Glu Leu Val Pro         355           #       360           #       365 ggc tca gtg cac ctc aac acc ccc gtc gct ga #a att gag cag tcg gca     1152 Gly Ser Val His Leu Asn Thr Pro Val Ala Gl #u Ile Glu Gln Ser Ala     370               #   375               #   380 tcc ggc tgt aca gta cga tcg gcc tcg ggc gc #c gtg ttc cga agc aaa     1200 Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Al #a Val Phe Arg Ser Lys 385                 3 #90                 3 #95                 4 #00 aag gtg gtg gtt tcg tta ccg aca acc ttg ta #t ccc acc ttg aca ttt     1248 Lys Val Val Val Ser Leu Pro Thr Thr Leu Ty #r Pro Thr Leu Thr Phe                 405   #               410   #               415 tca cca cct ctt ccc gcc gag aag caa gca tt #g gcg gaa aat tct atc     1296 Ser Pro Pro Leu Pro Ala Glu Lys Gln Ala Le #u Ala Glu Asn Ser Ile             420       #           425       #           430 ctg ggc tac tat agc aag ata gtc ttc gta tg #g gac aag ccg tgg tgg     1344 Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val Tr #p Asp Lys Pro Trp Trp         435           #       440           #       445 cgc gaa caa ggc ttc tcg ggc gtc ctc caa tc #g agc tgt gac ccc atc     1392 Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Se #r Ser Cys Asp Pro Ile     450               #   455               #   460 tca ttt gcc aga gat acc agc atc gac gtc ga #t cga caa tgg tcc att     1440 Ser Phe Ala Arg Asp Thr Ser Ile Asp Val As #p Arg Gln Trp Ser Ile 465                 4 #70                 4 #75                 4 #80 acc tgt ttc atg gtc gga gac ccg gga cgg aa #g tgg tcc caa cag tcc     1488 Thr Cys Phe Met Val Gly Asp Pro Gly Arg Ly #s Trp Ser Gln Gln Ser                 485   #               490   #               495 aag cag gta cga caa aag tct gtc tgg gac ca #a ctc cgc gca gcc tac     1536 Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gl #n Leu Arg Ala Ala Tyr             500       #           505       #           510 gag aac gcc ggg gcc caa gtc cca gag ccg gc #c aac gtg ctc gaa atc     1584 Glu Asn Ala Gly Ala Gln Val Pro Glu Pro Al #a Asn Val Leu Glu Ile         515           #       520           #       525 gag tgg tcg aag cag cag tat ttc caa gga gc #t ccg agc gcc gtc tat     1632 Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Al #a Pro Ser Ala Val Tyr     530               #   535               #   540 ggg ctg aac gat ctc atc aca ctg ggt tcg gc #g ctc aga acg ccg ttc     1680 Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Al #a Leu Arg Thr Pro Phe 545                 5 #50                 5 #55                 5 #60 aag agt gtt cat ttc gtt gga acg gag acg tc #t tta gtt tgg aaa ggg     1728 Lys Ser Val His Phe Val Gly Thr Glu Thr Se #r Leu Val Trp Lys Gly                 565   #               570   #               575 tat atg gaa ggg gcc ata cga tcg ggt caa cg #a ggt gct gca gaa gtt     1776 Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Ar #g Gly Ala Ala Glu Val             580       #           585       #           590 gtg gct agc ctg gtg cca gca gca tag     #                   #           1803 Val Ala Ser Leu Val Pro Ala Ala         595           #       600 <210> SEQ ID NO 23 <211> LENGTH: 600 <212> TYPE: PRT <213> ORGANISM: Exophiala spinifera <400> SEQUENCE: 23 Met Ala Leu Ala Pro Ser Tyr Ile Asn Pro Pr #o Asn Val Ala Ser Pro  1               5   #                10   #                15 Ala Gly Tyr Ser His Val Gly Val Gly Pro As #p Gly Gly Arg Tyr Val             20       #            25       #            30 Thr Ile Ala Gly Gln Ile Gly Gln Asp Ala Se #r Gly Val Thr Asp Pro         35           #        40           #        45 Ala Tyr Glu Lys Gln Val Ala Gln Ala Phe Al #a Asn Leu Arg Ala Cys     50               #    55               #    60 Leu Ala Ala Val Gly Ala Thr Ser Asn Asp Va #l Thr Lys Leu Asn Tyr 65                   #70                   #75                   #80 Tyr Ile Val Asp Tyr Ala Pro Ser Lys Leu Th #r Ala Ile Gly Asp Gly                 85   #                90   #                95 Leu Lys Ala Thr Phe Ala Leu Asp Arg Leu Pr #o Pro Cys Thr Leu Val             100       #           105       #           110 Pro Val Ser Ala Leu Ser Ser Pro Glu Tyr Le #u Phe Glu Val Asp Ala         115           #       120           #       125 Thr Ala Leu Val Pro Gly His Thr Thr Pro As #p Asn Val Ala Asp Val     130               #   135               #   140 Val Val Val Gly Ala Gly Leu Ser Gly Leu Gl #u Thr Ala Arg Lys Val 145                 1 #50                 1 #55                 1 #60 Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Gl #u Ala Met Asp Arg Val                 165   #               170   #               175 Gly Gly Lys Thr Leu Ser Val Gln Ser Gly Pr #o Gly Arg Thr Thr Ile             180       #           185       #           190 Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Se #r Asn Gln Ser Glu Val         195           #       200           #       205 Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gl #y Glu Leu Gln Arg Thr     210               #   215               #   220 Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gl #y Thr Thr Thr Thr Ala 225                 2 #30                 2 #35                 2 #40 Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Va #l Ala Ser Ala Leu Ala                 245   #               250   #               255 Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Gl #u Glu His Ser Leu Gln             260       #           265       #           270 Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Le #u Asp Ser Val Ser Phe         275           #       280           #       285 Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pr #o Ala Val Leu Gly Val     290               #   295               #   300 Ala Asn Gln Ile Thr Arg Ala Leu Leu Gly Va #l Glu Ala His Glu Ile 305                 3 #10                 3 #15                 3 #20 Ser Met Leu Phe Leu Thr Asp Tyr Ile Lys Se #r Ala Thr Gly Leu Ser                 325   #               330   #               335 Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gl #n Tyr Met Arg Cys Lys             340       #           345       #           350 Thr Gly Met Gln Ser Ile Cys His Ala Met Se #r Lys Glu Leu Val Pro         355           #       360           #       365 Gly Ser Val His Leu Asn Thr Pro Val Ala Gl #u Ile Glu Gln Ser Ala     370               #   375               #   380 Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Al #a Val Phe Arg Ser Lys 385                 3 #90                 3 #95                 4 #00 Lys Val Val Val Ser Leu Pro Thr Thr Leu Ty #r Pro Thr Leu Thr Phe                 405   #               410   #               415 Ser Pro Pro Leu Pro Ala Glu Lys Gln Ala Le #u Ala Glu Asn Ser Ile             420       #           425       #           430 Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val Tr #p Asp Lys Pro Trp Trp         435           #       440           #       445 Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Se #r Ser Cys Asp Pro Ile     450               #   455               #   460 Ser Phe Ala Arg Asp Thr Ser Ile Asp Val As #p Arg Gln Trp Ser Ile 465                 4 #70                 4 #75                 4 #80 Thr Cys Phe Met Val Gly Asp Pro Gly Arg Ly #s Trp Ser Gln Gln Ser                 485   #               490   #               495 Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gl #n Leu Arg Ala Ala Tyr             500       #           505       #           510 Glu Asn Ala Gly Ala Gln Val Pro Glu Pro Al #a Asn Val Leu Glu Ile         515           #       520           #       525 Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Al #a Pro Ser Ala Val Tyr     530               #   535               #   540 Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Al #a Leu Arg Thr Pro Phe 545                 5 #50                 5 #55                 5 #60 Lys Ser Val His Phe Val Gly Thr Glu Thr Se #r Leu Val Trp Lys Gly                 565   #               570   #               575 Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Ar #g Gly Ala Ala Glu Val             580       #           585       #           590 Val Ala Ser Leu Val Pro Ala Ala         595           #       600 <210> SEQ ID NO 24 <211> LENGTH: 3003 <212> TYPE: DNA <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Sequence is a barley a #lpha amylase signal       sequence: esp1 mat: an artificial  #spacer sequence and K:trAPAO <220> FEATURE: <221> NAME/KEY: sig_peptide <222> LOCATION: (1)...(72) <223> OTHER INFORMATION: Barley alpha amylase sign #al sequence <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (73)...(1575) <223> OTHER INFORMATION: esp1 mat <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1576)...(1611) <223> OTHER INFORMATION: spacer sequence <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1612)...(3000) <223> OTHER INFORMATION: K:trAPAO <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(3000) <221> NAME/KEY: misc_feature <222> LOCATION: (1612)...(1614) <223> OTHER INFORMATION: Extra lysine <400> SEQUENCE: 24 atg gcc aac aag cac ctg agc ctc tcc ctc tt #c ctc gtg ctc ctc ggc       48 Met Ala Asn Lys His Leu Ser Leu Ser Leu Ph #e Leu Val Leu Leu Gly                 -20   #               -15   #               -10 ctc tcc gcc tcc ctc gcc agc ggc gct cct ac #t gtc aag att gat gct       96 Leu Ser Ala Ser Leu Ala Ser Gly Ala Pro Th #r Val Lys Ile Asp Ala              -5      #            1       #         5 ggg atg gtg gtc ggc acg act act act gtc cc #c ggc acc act gcg acc      144 Gly Met Val Val Gly Thr Thr Thr Thr Val Pr #o Gly Thr Thr Ala Thr      10              #     15              #     20 gtc agc gag ttc ttg ggc gtt cct ttt gcc gc #c tct ccg aca cga ttt      192 Val Ser Glu Phe Leu Gly Val Pro Phe Ala Al #a Ser Pro Thr Arg Phe  25                  # 30                  # 35                  # 40 gcg cct cct act cgt ccc gtg cct tgg tca ac #g cct ttg caa gcc act      240 Ala Pro Pro Thr Arg Pro Val Pro Trp Ser Th #r Pro Leu Gln Ala Thr                  45  #                 50  #                 55 gca tat ggt cca gca tgc cct caa caa ttc aa #t tac ccc gaa gaa ctc      288 Ala Tyr Gly Pro Ala Cys Pro Gln Gln Phe As #n Tyr Pro Glu Glu Leu              60      #             65      #             70 cgt gag att acg atg gcc tgg ttc aat aca cc #g ccc ccg tca gct ggt      336 Arg Glu Ile Thr Met Ala Trp Phe Asn Thr Pr #o Pro Pro Ser Ala Gly          75          #         80          #         85 gaa agt gag gac tgc ctg aac ctc aac atc ta #c gtc cca gga act gag      384 Glu Ser Glu Asp Cys Leu Asn Leu Asn Ile Ty #r Val Pro Gly Thr Glu      90              #     95              #    100 aac aca aac aaa gcc gtc atg gtt tgg ata ta #c ggt gga gcg ctg gaa      432 Asn Thr Asn Lys Ala Val Met Val Trp Ile Ty #r Gly Gly Ala Leu Glu 105                 1 #10                 1 #15                 1 #20 tat ggt tgg aat tca ttc cac ctt tac gac gg #g gct agt ttc gca gcc      480 Tyr Gly Trp Asn Ser Phe His Leu Tyr Asp Gl #y Ala Ser Phe Ala Ala                 125   #               130   #               135 aat cag gat gtc atc gcc gtg acc atc aac ta #c aga acg aac att ctg      528 Asn Gln Asp Val Ile Ala Val Thr Ile Asn Ty #r Arg Thr Asn Ile Leu             140       #           145       #           150 ggg ttc cct gct gcc cct cag ctt cca ata ac #a cag cga aat ctg ggg      576 Gly Phe Pro Ala Ala Pro Gln Leu Pro Ile Th #r Gln Arg Asn Leu Gly         155           #       160           #       165 ttc cta gac caa agg ttt gct ttg gat tgg gt #a cag cgg aac atc gca      624 Phe Leu Asp Gln Arg Phe Ala Leu Asp Trp Va #l Gln Arg Asn Ile Ala     170               #   175               #   180 gcc ttt ggc ggt gat cct cga aag gtc aca at #a ttt ggg cag agt gcg      672 Ala Phe Gly Gly Asp Pro Arg Lys Val Thr Il #e Phe Gly Gln Ser Ala 185                 1 #90                 1 #95                 2 #00 ggg ggc aga agt gtc gac gtc ctc ttg acg tc #t atg cca cac aac cca      720 Gly Gly Arg Ser Val Asp Val Leu Leu Thr Se #r Met Pro His Asn Pro                 205   #               210   #               215 ccc ttc cga gca gca atc atg gag tcc ggt gt #g gct aac tac aac ttc      768 Pro Phe Arg Ala Ala Ile Met Glu Ser Gly Va #l Ala Asn Tyr Asn Phe             220       #           225       #           230 ccc aag gga gat ttg tcc gaa cct tgg aac ac #c act gtt caa gct ctc      816 Pro Lys Gly Asp Leu Ser Glu Pro Trp Asn Th #r Thr Val Gln Ala Leu         235           #       240           #       245 aac tgt acc acc agt atc gac atc ttg agt tg #t atg aga aga gtc gat      864 Asn Cys Thr Thr Ser Ile Asp Ile Leu Ser Cy #s Met Arg Arg Val Asp     250               #   255               #   260 ctc gcc act ctg atg aac acg atc gag caa ct #c gga ctt ggg ttt gag      912 Leu Ala Thr Leu Met Asn Thr Ile Glu Gln Le #u Gly Leu Gly Phe Glu 265                 2 #70                 2 #75                 2 #80 tac acg ttg gac aac gta acg gct gtg tac cg #t tct gaa acg gct cgc      960 Tyr Thr Leu Asp Asn Val Thr Ala Val Tyr Ar #g Ser Glu Thr Ala Arg                 285   #               290   #               295 acg act ggt gac att gct cgt gta cct gtt ct #c gtc ggg acg gtg gcc     1008 Thr Thr Gly Asp Ile Ala Arg Val Pro Val Le #u Val Gly Thr Val Ala             300       #           305       #           310 aac gac gga ctt ctc ttt gtc ctc ggg gag aa #t gac acc caa gca tat     1056 Asn Asp Gly Leu Leu Phe Val Leu Gly Glu As #n Asp Thr Gln Ala Tyr         315           #       320           #       325 ctc gag gag gca atc ccg aat cag ccc gac ct #t tac cag act ctc ctt     1104 Leu Glu Glu Ala Ile Pro Asn Gln Pro Asp Le #u Tyr Gln Thr Leu Leu     330               #   335               #   340 gga gca tat ccc att gga tcc cca ggg atc gg #a tcg cct caa gat cag     1152 Gly Ala Tyr Pro Ile Gly Ser Pro Gly Ile Gl #y Ser Pro Gln Asp Gln 345                 3 #50                 3 #55                 3 #60 att gcc gcc att gag acc gag gta aga ttc ca #g tgt cct tct gcc atc     1200 Ile Ala Ala Ile Glu Thr Glu Val Arg Phe Gl #n Cys Pro Ser Ala Ile                 365   #               370   #               375 gtg gct cag gac tcc cgg aat cgg ggt atc cc #t tct tgg cgc tac tac     1248 Val Ala Gln Asp Ser Arg Asn Arg Gly Ile Pr #o Ser Trp Arg Tyr Tyr             380       #           385       #           390 tac aat gcg acc ttt gag aat ctg gag ctt tt #c cct ggg tcc gaa gtg     1296 Tyr Asn Ala Thr Phe Glu Asn Leu Glu Leu Ph #e Pro Gly Ser Glu Val         395           #       400           #       405 tac cac agc tct gaa gtc ggg atg gtg ttt gg #c acg tat cct gtc gca     1344 Tyr His Ser Ser Glu Val Gly Met Val Phe Gl #y Thr Tyr Pro Val Ala     410               #   415               #   420 agt gcg acc gcc ttg gag gcc cag acg agc aa #a tac atg cag ggt gcc     1392 Ser Ala Thr Ala Leu Glu Ala Gln Thr Ser Ly #s Tyr Met Gln Gly Ala 425                 4 #30                 4 #35                 4 #40 tgg gcg gcc ttt gcc aaa aac ccc atg aat gg #g cct ggg tgg aaa caa     1440 Trp Ala Ala Phe Ala Lys Asn Pro Met Asn Gl #y Pro Gly Trp Lys Gln                 445   #               450   #               455 gtg ccg aat gtc gcg gcg ctt ggc tca cca gg #c aaa gcc atc cag gtt     1488 Val Pro Asn Val Ala Ala Leu Gly Ser Pro Gl #y Lys Ala Ile Gln Val             460       #           465       #           470 gac gtc tct cca gcg aca ata gac caa cga tg #t gcc ttg tac acg cgt     1536 Asp Val Ser Pro Ala Thr Ile Asp Gln Arg Cy #s Ala Leu Tyr Thr Arg         475           #       480           #       485 tat tat act gag ttg ggc aca atc gcg ccg ag #g aca ttt ggc gga ggc     1584 Tyr Tyr Thr Glu Leu Gly Thr Ile Ala Pro Ar #g Thr Phe Gly Gly Gly     490               #   495               #   500 agc ggc gga ggc agc ggc gga ggc agc aaa ga #c aac gtt gcg gac gtg     1632 Ser Gly Gly Gly Ser Gly Gly Gly Ser Lys As #p Asn Val Ala Asp Val 505                 5 #10                 5 #15                 5 #20 gta gtg gtg ggc gct ggc ttg agc ggt ttg ga #g acg gca cgc aaa gtc     1680 Val Val Val Gly Ala Gly Leu Ser Gly Leu Gl #u Thr Ala Arg Lys Val                 525   #               530   #               535 cag gcc gcc ggt ctg tcc tgc ctc gtt ctt ga #g gcg atg gat cgt gta     1728 Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Gl #u Ala Met Asp Arg Val             540       #           545       #           550 ggg gga aag act ctg agc gta caa tcg ggt cc #c ggc agg acg act atc     1776 Gly Gly Lys Thr Leu Ser Val Gln Ser Gly Pr #o Gly Arg Thr Thr Ile         555           #       560           #       565 aac gac ctc ggc gct gcg tgg atc aat gac ag #c aac caa agc gaa gta     1824 Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Se #r Asn Gln Ser Glu Val     570               #   575               #   580 tcc aga ttg ttt gaa aga ttt cat ttg gag gg #c gag ctc cag agg acg     1872 Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gl #y Glu Leu Gln Arg Thr 585                 5 #90                 5 #95                 6 #00 act gga aat tca atc cat caa gca caa gac gg #t aca acc act aca gct     1920 Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gl #y Thr Thr Thr Thr Ala                 605   #               610   #               615 cct tat ggt gac tcc ttg ctg agc gag gag gt #t gca agt gca ctt gcg     1968 Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Va #l Ala Ser Ala Leu Ala             620       #           625       #           630 gaa ctc ctc ccc gta tgg tct cag ctg atc ga #a gag cat agc ctt caa     2016 Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Gl #u Glu His Ser Leu Gln         635           #       640           #       645 gac ctc aag gcg agc cct cag gcg aag cgg ct #c gac agt gtg agc ttc     2064 Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Le #u Asp Ser Val Ser Phe     650               #   655               #   660 gcg cac tac tgt gag aag gaa cta aac ttg cc #t gct gtt ctc ggc gta     2112 Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pr #o Ala Val Leu Gly Val 665                 6 #70                 6 #75                 6 #80 gca aac cag atc aca cgc gct ctg ctc ggt gt #g gaa gcc cac gag atc     2160 Ala Asn Gln Ile Thr Arg Ala Leu Leu Gly Va #l Glu Ala His Glu Ile                 685   #               690   #               695 agc atg ctt ttt ctc acc gac tac atc aag ag #t gcc acc ggt ctc agt     2208 Ser Met Leu Phe Leu Thr Asp Tyr Ile Lys Se #r Ala Thr Gly Leu Ser             700       #           705       #           710 aat att ttc tcg gac aag aaa gac ggc ggg ca #g tat atg cga tgc aaa     2256 Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gl #n Tyr Met Arg Cys Lys         715           #       720           #       725 aca ggt atg cag tcg att tgc cat gcc atg tc #a aag gaa ctt gtt cca     2304 Thr Gly Met Gln Ser Ile Cys His Ala Met Se #r Lys Glu Leu Val Pro     730               #   735               #   740 ggc tca gtg cac ctc aac acc ccc gtc gct ga #a att gag cag tcg gca     2352 Gly Ser Val His Leu Asn Thr Pro Val Ala Gl #u Ile Glu Gln Ser Ala 745                 7 #50                 7 #55                 7 #60 tcc ggc tgt aca gta cga tcg gcc tcg ggc gc #c gtg ttc cga agc aaa     2400 Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Al #a Val Phe Arg Ser Lys                 765   #               770   #               775 aag gtg gtg gtt tcg tta ccg aca acc ttg ta #t ccc acc ttg aca ttt     2448 Lys Val Val Val Ser Leu Pro Thr Thr Leu Ty #r Pro Thr Leu Thr Phe             780       #           785       #           790 tca cca cct ctt ccc gcc gag aag caa gca tt #g gcg gaa aat tct atc     2496 Ser Pro Pro Leu Pro Ala Glu Lys Gln Ala Le #u Ala Glu Asn Ser Ile         795           #       800           #       805 ctg ggc tac tat agc aag ata gtc ttc gta tg #g gac aag ccg tgg tgg     2544 Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val Tr #p Asp Lys Pro Trp Trp     810               #   815               #   820 cgc gaa caa ggc ttc tcg ggc gtc ctc caa tc #g agc tgt gac ccc atc     2592 Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Se #r Ser Cys Asp Pro Ile 825                 8 #30                 8 #35                 8 #40 tca ttt gcc aga gat acc agc atc gac gtc ga #t cga caa tgg tcc att     2640 Ser Phe Ala Arg Asp Thr Ser Ile Asp Val As #p Arg Gln Trp Ser Ile                 845   #               850   #               855 acc tgt ttc atg gtc gga gac ccg gga cgg aa #g tgg tcc caa cag tcc     2688 Thr Cys Phe Met Val Gly Asp Pro Gly Arg Ly #s Trp Ser Gln Gln Ser             860       #           865       #           870 aag cag gta cga caa aag tct gtc tgg gac ca #a ctc cgc gca gcc tac     2736 Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gl #n Leu Arg Ala Ala Tyr         875           #       880           #       885 gag aac gcc ggg gcc caa gtc cca gag ccg gc #c aac gtg ctc gaa atc     2784 Glu Asn Ala Gly Ala Gln Val Pro Glu Pro Al #a Asn Val Leu Glu Ile     890               #   895               #   900 gag tgg tcg aag cag cag tat ttc caa gga gc #t ccg agc gcc gtc tat     2832 Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Al #a Pro Ser Ala Val Tyr 905                 9 #10                 9 #15                 9 #20 ggg ctg aac gat ctc atc aca ctg ggt tcg gc #g ctc aga acg ccg ttc     2880 Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Al #a Leu Arg Thr Pro Phe                 925   #               930   #               935 aag agt gtt cat ttc gtt gga acg gag acg tc #t tta gtt tgg aaa ggg     2928 Lys Ser Val His Phe Val Gly Thr Glu Thr Se #r Leu Val Trp Lys Gly             940       #           945       #           950 tat atg gaa ggg gcc ata cga tcg ggt caa cg #a ggt gct gca gaa gtt     2976 Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Ar #g Gly Ala Ala Glu Val         955           #       960           #       965 gtg gct agc ctg gtg cca gca gca tag     #                   #           3003 Val Ala Ser Leu Val Pro Ala Ala     970               #   975 <210> SEQ ID NO 25 <211> LENGTH: 1000 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: SIGNAL <222> LOCATION: (1)...(24) <400> SEQUENCE: 25 Met Ala Asn Lys His Leu Ser Leu Ser Leu Ph #e Leu Val Leu Leu Gly                 -20   #               -15   #               -10 Leu Ser Ala Ser Leu Ala Ser Gly Ala Pro Th #r Val Lys Ile Asp Ala             -5       #             1      #          5 Gly Met Val Val Gly Thr Thr Thr Thr Val Pr #o Gly Thr Thr Ala Thr     10               #    15               #    20 Val Ser Glu Phe Leu Gly Val Pro Phe Ala Al #a Ser Pro Thr Arg Phe 25                   #30                   #35                   #40 Ala Pro Pro Thr Arg Pro Val Pro Trp Ser Th #r Pro Leu Gln Ala Thr                 45   #                50   #                55 Ala Tyr Gly Pro Ala Cys Pro Gln Gln Phe As #n Tyr Pro Glu Glu Leu             60       #            65       #            70 Arg Glu Ile Thr Met Ala Trp Phe Asn Thr Pr #o Pro Pro Ser Ala Gly         75           #        80           #        85 Glu Ser Glu Asp Cys Leu Asn Leu Asn Ile Ty #r Val Pro Gly Thr Glu     90               #    95               #    100 Asn Thr Asn Lys Ala Val Met Val Trp Ile Ty #r Gly Gly Ala Leu Glu 105                 1 #10                 1 #15                 1 #20 Tyr Gly Trp Asn Ser Phe His Leu Tyr Asp Gl #y Ala Ser Phe Ala Ala                 125   #               130   #               135 Asn Gln Asp Val Ile Ala Val Thr Ile Asn Ty #r Arg Thr Asn Ile Leu             140       #           145       #           150 Gly Phe Pro Ala Ala Pro Gln Leu Pro Ile Th #r Gln Arg Asn Leu Gly         155           #       160           #       165 Phe Leu Asp Gln Arg Phe Ala Leu Asp Trp Va #l Gln Arg Asn Ile Ala     170               #   175               #   180 Ala Phe Gly Gly Asp Pro Arg Lys Val Thr Il #e Phe Gly Gln Ser Ala 185                 1 #90                 1 #95                 2 #00 Gly Gly Arg Ser Val Asp Val Leu Leu Thr Se #r Met Pro His Asn Pro                 205   #               210   #               215 Pro Phe Arg Ala Ala Ile Met Glu Ser Gly Va #l Ala Asn Tyr Asn Phe             220       #           225       #           230 Pro Lys Gly Asp Leu Ser Glu Pro Trp Asn Th #r Thr Val Gln Ala Leu         235           #       240           #       245 Asn Cys Thr Thr Ser Ile Asp Ile Leu Ser Cy #s Met Arg Arg Val Asp     250               #   255               #   260 Leu Ala Thr Leu Met Asn Thr Ile Glu Gln Le #u Gly Leu Gly Phe Glu 265                 2 #70                 2 #75                 2 #80 Tyr Thr Leu Asp Asn Val Thr Ala Val Tyr Ar #g Ser Glu Thr Ala Arg                 285   #               290   #               295 Thr Thr Gly Asp Ile Ala Arg Val Pro Val Le #u Val Gly Thr Val Ala             300       #           305       #           310 Asn Asp Gly Leu Leu Phe Val Leu Gly Glu As #n Asp Thr Gln Ala Tyr         315           #       320           #       325 Leu Glu Glu Ala Ile Pro Asn Gln Pro Asp Le #u Tyr Gln Thr Leu Leu     330               #   335               #   340 Gly Ala Tyr Pro Ile Gly Ser Pro Gly Ile Gl #y Ser Pro Gln Asp Gln 345                 3 #50                 3 #55                 3 #60 Ile Ala Ala Ile Glu Thr Glu Val Arg Phe Gl #n Cys Pro Ser Ala Ile                 365   #               370   #               375 Val Ala Gln Asp Ser Arg Asn Arg Gly Ile Pr #o Ser Trp Arg Tyr Tyr             380       #           385       #           390 Tyr Asn Ala Thr Phe Glu Asn Leu Glu Leu Ph #e Pro Gly Ser Glu Val         395           #       400           #       405 Tyr His Ser Ser Glu Val Gly Met Val Phe Gl #y Thr Tyr Pro Val Ala     410               #   415               #   420 Ser Ala Thr Ala Leu Glu Ala Gln Thr Ser Ly #s Tyr Met Gln Gly Ala 425                 4 #30                 4 #35                 4 #40 Trp Ala Ala Phe Ala Lys Asn Pro Met Asn Gl #y Pro Gly Trp Lys Gln                 445   #               450   #               455 Val Pro Asn Val Ala Ala Leu Gly Ser Pro Gl #y Lys Ala Ile Gln Val             460       #           465       #           470 Asp Val Ser Pro Ala Thr Ile Asp Gln Arg Cy #s Ala Leu Tyr Thr Arg         475           #       480           #       485 Tyr Tyr Thr Glu Leu Gly Thr Ile Ala Pro Ar #g Thr Phe Gly Gly Gly     490               #   495               #   500 Ser Gly Gly Gly Ser Gly Gly Gly Ser Lys As #p Asn Val Ala Asp Val 505                 5 #10                 5 #15                 5 #20 Val Val Val Gly Ala Gly Leu Ser Gly Leu Gl #u Thr Ala Arg Lys Val                 525   #               530   #               535 Gln Ala Ala Gly Leu Ser Cys Leu Val Leu Gl #u Ala Met Asp Arg Val             540       #           545       #           550 Gly Gly Lys Thr Leu Ser Val Gln Ser Gly Pr #o Gly Arg Thr Thr Ile         555           #       560           #       565 Asn Asp Leu Gly Ala Ala Trp Ile Asn Asp Se #r Asn Gln Ser Glu Val     570               #   575               #   580 Ser Arg Leu Phe Glu Arg Phe His Leu Glu Gl #y Glu Leu Gln Arg Thr 585                 5 #90                 5 #95                 6 #00 Thr Gly Asn Ser Ile His Gln Ala Gln Asp Gl #y Thr Thr Thr Thr Ala                 605   #               610   #               615 Pro Tyr Gly Asp Ser Leu Leu Ser Glu Glu Va #l Ala Ser Ala Leu Ala             620       #           625       #           630 Glu Leu Leu Pro Val Trp Ser Gln Leu Ile Gl #u Glu His Ser Leu Gln         635           #       640           #       645 Asp Leu Lys Ala Ser Pro Gln Ala Lys Arg Le #u Asp Ser Val Ser Phe     650               #   655               #   660 Ala His Tyr Cys Glu Lys Glu Leu Asn Leu Pr #o Ala Val Leu Gly Val 665                 6 #70                 6 #75                 6 #80 Ala Asn Gln Ile Thr Arg Ala Leu Leu Gly Va #l Glu Ala His Glu Ile                 685   #               690   #               695 Ser Met Leu Phe Leu Thr Asp Tyr Ile Lys Se #r Ala Thr Gly Leu Ser             700       #           705       #           710 Asn Ile Phe Ser Asp Lys Lys Asp Gly Gly Gl #n Tyr Met Arg Cys Lys         715           #       720           #       725 Thr Gly Met Gln Ser Ile Cys His Ala Met Se #r Lys Glu Leu Val Pro     730               #   735               #   740 Gly Ser Val His Leu Asn Thr Pro Val Ala Gl #u Ile Glu Gln Ser Ala 745                 7 #50                 7 #55                 7 #60 Ser Gly Cys Thr Val Arg Ser Ala Ser Gly Al #a Val Phe Arg Ser Lys                 765   #               770   #               775 Lys Val Val Val Ser Leu Pro Thr Thr Leu Ty #r Pro Thr Leu Thr Phe             780       #           785       #           790 Ser Pro Pro Leu Pro Ala Glu Lys Gln Ala Le #u Ala Glu Asn Ser Ile         795           #       800           #       805 Leu Gly Tyr Tyr Ser Lys Ile Val Phe Val Tr #p Asp Lys Pro Trp Trp     810               #   815               #   820 Arg Glu Gln Gly Phe Ser Gly Val Leu Gln Se #r Ser Cys Asp Pro Ile 825                 8 #30                 8 #35                 8 #40 Ser Phe Ala Arg Asp Thr Ser Ile Asp Val As #p Arg Gln Trp Ser Ile                 845   #               850   #               855 Thr Cys Phe Met Val Gly Asp Pro Gly Arg Ly #s Trp Ser Gln Gln Ser             860       #           865       #           870 Lys Gln Val Arg Gln Lys Ser Val Trp Asp Gl #n Leu Arg Ala Ala Tyr         875           #       880           #       885 Glu Asn Ala Gly Ala Gln Val Pro Glu Pro Al #a Asn Val Leu Glu Ile     890               #   895               #   900 Glu Trp Ser Lys Gln Gln Tyr Phe Gln Gly Al #a Pro Ser Ala Val Tyr 905                 9 #10                 9 #15                 9 #20 Gly Leu Asn Asp Leu Ile Thr Leu Gly Ser Al #a Leu Arg Thr Pro Phe                 925   #               930   #               935 Lys Ser Val His Phe Val Gly Thr Glu Thr Se #r Leu Val Trp Lys Gly             940       #           945       #           950 Tyr Met Glu Gly Ala Ile Arg Ser Gly Gln Ar #g Gly Ala Ala Glu Val         955           #       960           #       965 Val Ala Ser Leu Val Pro Ala Ala     970               #   975 <210> SEQ ID NO 26 <211> LENGTH: 2976 <212> TYPE: DNA <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Barley alpha amylase sign #al sequence: BEST1       mature: artificial spacer: and K:trA #PAO.  For plant expression. <220> FEATURE: <221> NAME/KEY: sig_peptide <222> LOCATION: (1)...(72) <223> OTHER INFORMATION: Barley alpha amylase sign #al sequence. <220> FEATURE: <221> NAME/KEY: mat_peptide <222> LOCATION: (73)...(1545) <223> OTHER INFORMATION: BEST1 mature <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1546)...(1584) <223> OTHER INFORMATION: Artificial spacer sequence <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1585)...(2973) <223> OTHER INFORMATION: K:trAPAO <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(2973) <221> NAME/KEY: misc_feature <222> LOCATION: (1585)...(1587) <223> OTHER INFORMATION: Extra lysine <400> SEQUENCE: 26 atg gcc aac aag cac ctg agc ctc tcc ctc tt #c ctc gtg ctc ctc ggc       48 Met Ala Asn Lys His Leu Ser Leu Ser Leu Ph #e Leu Val Leu Leu Gly                 -20   #               -15   #               -10 ctc tcc gcc tcc ctc gcc agc ggc acg gat tt #t ccg gtc cgc agg acc       96 Leu Ser Ala Ser Leu Ala Ser Gly Thr Asp Ph #e Pro Val Arg Arg Thr              -5      #            1       #         5 gat ctg ggc cag gtt cag gga ctg gcc ggg ga #c gtg atg agc ttt cgc      144 Asp Leu Gly Gln Val Gln Gly Leu Ala Gly As #p Val Met Ser Phe Arg      10              #     15              #     20 gga ata ccc tat gca gcg ccg ccg gtg ggc gg #g ctg cgt tgg aag ccg      192 Gly Ile Pro Tyr Ala Ala Pro Pro Val Gly Gl #y Leu Arg Trp Lys Pro  25                  # 30                  # 35                  # 40 ccc caa cac gcc cgg ccc tgg gcg ggc gtt cg #c ccc gcc acc caa ttt      240 Pro Gln His Ala Arg Pro Trp Ala Gly Val Ar #g Pro Ala Thr Gln Phe                  45  #                 50  #                 55 ggc tcc gac tgc ttc ggc gcg gcc tat ctt cg #c aaa ggc agc ctc gcc      288 Gly Ser Asp Cys Phe Gly Ala Ala Tyr Leu Ar #g Lys Gly Ser Leu Ala              60      #             65      #             70 ccc ggc gtg agc gag gac tgt ctt tac ctc aa #c gta tgg gcg ccg tca      336 Pro Gly Val Ser Glu Asp Cys Leu Tyr Leu As #n Val Trp Ala Pro Ser          75          #         80          #         85 ggc gct aaa ccc ggc cag tac ccc gtc atg gt #c tgg gtc tac ggc ggc      384 Gly Ala Lys Pro Gly Gln Tyr Pro Val Met Va #l Trp Val Tyr Gly Gly      90              #     95              #    100 ggc ttc gcc ggc ggc acg gcc gcc atg ccc ta #c tac gac ggc gag gcg      432 Gly Phe Ala Gly Gly Thr Ala Ala Met Pro Ty #r Tyr Asp Gly Glu Ala 105                 1 #10                 1 #15                 1 #20 ctt gcg cga cag ggc gtc gtc gtg gtg acg tt #t aac tat cgg acg aac      480 Leu Ala Arg Gln Gly Val Val Val Val Thr Ph #e Asn Tyr Arg Thr Asn                 125   #               130   #               135 atc ctg ggc ttt ttc gcc cat cct ggt ctc tc #g cgc gag agc ccc acc      528 Ile Leu Gly Phe Phe Ala His Pro Gly Leu Se #r Arg Glu Ser Pro Thr             140       #           145       #           150 gga act tcg ggc aac tac ggc cta ctc gac at #t ctc gcc gct ctt cgg      576 Gly Thr Ser Gly Asn Tyr Gly Leu Leu Asp Il #e Leu Ala Ala Leu Arg         155           #       160           #       165 tgg gtg cag agc aac gcc cgc gcc ttc gga gg #g gac ccc ggc cga gtg      624 Trp Val Gln Ser Asn Ala Arg Ala Phe Gly Gl #y Asp Pro Gly Arg Val     170               #   175               #   180 acg gtc ttt ggt gaa tcg gcc gga gcg agc gc #g atc gga ctt ctg ctc      672 Thr Val Phe Gly Glu Ser Ala Gly Ala Ser Al #a Ile Gly Leu Leu Leu 185                 1 #90                 1 #95                 2 #00 acc tcg ccg ctg agc aag ggt ctc ttc cgt gg #c gct atc ctc gaa agt      720 Thr Ser Pro Leu Ser Lys Gly Leu Phe Arg Gl #y Ala Ile Leu Glu Ser                 205   #               210   #               215 cca ggg ctg acg cga ccg ctc gcg acg ctc gc #c gac agc gcc gcc tcg      768 Pro Gly Leu Thr Arg Pro Leu Ala Thr Leu Al #a Asp Ser Ala Ala Ser             220       #           225       #           230 ggc gag cgc ctc gac gcc gat ctt tcg cga ct #g cgc tcg acc gac cca      816 Gly Glu Arg Leu Asp Ala Asp Leu Ser Arg Le #u Arg Ser Thr Asp Pro         235           #       240           #       245 gcc acc ctg atg gcg cgc gcc gac gcg gcc cg #c ccg gca tcg cgg gac      864 Ala Thr Leu Met Ala Arg Ala Asp Ala Ala Ar #g Pro Ala Ser Arg Asp     250               #   255               #   260 ctg cgc agg ccg cgt ccg acc gga ccg atc gt #c gat ggc cat gtg ctg      912 Leu Arg Arg Pro Arg Pro Thr Gly Pro Ile Va #l Asp Gly His Val Leu 265                 2 #70                 2 #75                 2 #80 ccg cag acc gac agc gcg gcg atc gcg gcg gg #g cag ctg gcg ccg gtt      960 Pro Gln Thr Asp Ser Ala Ala Ile Ala Ala Gl #y Gln Leu Ala Pro Val                 285   #               290   #               295 cgg gtc ctg atc gga acc aat gcc gac gaa gg #c cgc gcc ttc ctc ggg     1008 Arg Val Leu Ile Gly Thr Asn Ala Asp Glu Gl #y Arg Ala Phe Leu Gly             300       #           305       #           310 cgc gcg ccg atg gag acg cca gcg gac tac ca #a gcc tat ctg gag gcg     1056 Arg Ala Pro Met Glu Thr Pro Ala Asp Tyr Gl #n Ala Tyr Leu Glu Ala         315           #       320           #       325 cag ttt ggc gac caa gcc gcc gcc gtg gcg gc #g tgc tat ccc ctc gac     1104 Gln Phe Gly Asp Gln Ala Ala Ala Val Ala Al #a Cys Tyr Pro Leu Asp     330               #   335               #   340 ggc cgg gcc acg ccc aag gaa atg gtc gcg cg #c atc ttc ggc gac aat     1152 Gly Arg Ala Thr Pro Lys Glu Met Val Ala Ar #g Ile Phe Gly Asp Asn 345                 3 #50                 3 #55                 3 #60 cag ttc aat cgg ggg gtc tcg gcc ttc tcg ga #a gcg ctt gtg cgc cag     1200 Gln Phe Asn Arg Gly Val Ser Ala Phe Ser Gl #u Ala Leu Val Arg Gln                 365   #               370   #               375 ggc gcg ccc gtg tgg cgt tat cag ttc aac gg #t aat acc gag ggt gga     1248 Gly Ala Pro Val Trp Arg Tyr Gln Phe Asn Gl #y Asn Thr Glu Gly Gly             380       #           385       #           390 aga gcg ccg gct acc cac gga gcc gaa att cc #c tac gtt ttc ggg gtg     1296 Arg Ala Pro Ala Thr His Gly Ala Glu Ile Pr #o Tyr Val Phe Gly Val         395           #       400           #       405 ttc aag ctc gac gag ttg ggt ctg ttc gat tg #g ccg ccc gag ggg ccc     1344 Phe Lys Leu Asp Glu Leu Gly Leu Phe Asp Tr #p Pro Pro Glu Gly Pro     410               #   415               #   420 acg ccc gcc gac cgt gcg ctg ggc caa ctg at #g tcc tcc gcc tgg gtc     1392 Thr Pro Ala Asp Arg Ala Leu Gly Gln Leu Me #t Ser Ser Ala Trp Val 425                 4 #30                 4 #35                 4 #40 cgg ttc gcc aag aat ggc gac ccc gcc ggg ga #c gcc ctt acc tgg cct     1440 Arg Phe Ala Lys Asn Gly Asp Pro Ala Gly As #p Ala Leu Thr Trp Pro                 445   #               450   #               455 gcc tat tct acg ggc aag tcg acc atg aca tt #c ggt ccc gag ggc cgc     1488 Ala Tyr Ser Thr Gly Lys Ser Thr Met Thr Ph #e Gly Pro Glu Gly Arg             460       #           465       #           470 gcg gcg gtg gtg tcg ccc gga cct tcc atc cc #c cct tgc gcg gat ggc     1536 Ala Ala Val Val Ser Pro Gly Pro Ser Ile Pr #o Pro Cys Ala Asp Gly         475           #       480           #       485 gcc aag gcg ggg ggc gga ggc agc ggc gga gg #c agc ggc gga ggc agc     1584 Ala Lys Ala Gly Gly Gly Gly Ser Gly Gly Gl #y Ser Gly Gly Gly Ser     490               #   495               #   500 aaa gac aac gtt gcg gac gtg gta gtg gtg gg #c gct ggc ttg agc ggt     1632 Lys Asp Asn Val Ala Asp Val Val Val Val Gl #y Ala Gly Leu Ser Gly 505                 5 #10                 5 #15                 5 #20 ttg gag acg gca cgc aaa gtc cag gcc gcc gg #t ctg tcc tgc ctc gtt     1680 Leu Glu Thr Ala Arg Lys Val Gln Ala Ala Gl #y Leu Ser Cys Leu Val                 525   #               530   #               535 ctt gag gcg atg gat cgt gta ggg gga aag ac #t ctg agc gta caa tcg     1728 Leu Glu Ala Met Asp Arg Val Gly Gly Lys Th #r Leu Ser Val Gln Ser             540       #           545       #           550 ggt ccc ggc agg acg act atc aac gac ctc gg #c gct gcg tgg atc aat     1776 Gly Pro Gly Arg Thr Thr Ile Asn Asp Leu Gl #y Ala Ala Trp Ile Asn         555           #       560           #       565 gac agc aac caa agc gaa gta tcc aga ttg tt #t gaa aga ttt cat ttg     1824 Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Ph #e Glu Arg Phe His Leu     570               #   575               #   580 gag ggc gag ctc cag agg acg act gga aat tc #a atc cat caa gca caa     1872 Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Se #r Ile His Gln Ala Gln 585                 5 #90                 5 #95                 6 #00 gac ggt aca acc act aca gct cct tat ggt ga #c tcc ttg ctg agc gag     1920 Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly As #p Ser Leu Leu Ser Glu                 605   #               610   #               615 gag gtt gca agt gca ctt gcg gaa ctc ctc cc #c gta tgg tct cag ctg     1968 Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pr #o Val Trp Ser Gln Leu             620       #           625       #           630 atc gaa gag cat agc ctt caa gac ctc aag gc #g agc cct cag gcg aag     2016 Ile Glu Glu His Ser Leu Gln Asp Leu Lys Al #a Ser Pro Gln Ala Lys         635           #       640           #       645 cgg ctc gac agt gtg agc ttc gcg cac tac tg #t gag aag gaa cta aac     2064 Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cy #s Glu Lys Glu Leu Asn     650               #   655               #   660 ttg cct gct gtt ctc ggc gta gca aac cag at #c aca cgc gct ctg ctc     2112 Leu Pro Ala Val Leu Gly Val Ala Asn Gln Il #e Thr Arg Ala Leu Leu 665                 6 #70                 6 #75                 6 #80 ggt gtg gaa gcc cac gag atc agc atg ctt tt #t ctc acc gac tac atc     2160 Gly Val Glu Ala His Glu Ile Ser Met Leu Ph #e Leu Thr Asp Tyr Ile                 685   #               690   #               695 aag agt gcc acc ggt ctc agt aat att ttc tc #g gac aag aaa gac ggc     2208 Lys Ser Ala Thr Gly Leu Ser Asn Ile Phe Se #r Asp Lys Lys Asp Gly             700       #           705       #           710 ggg cag tat atg cga tgc aaa aca ggt atg ca #g tcg att tgc cat gcc     2256 Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gl #n Ser Ile Cys His Ala         715           #       720           #       725 atg tca aag gaa ctt gtt cca ggc tca gtg ca #c ctc aac acc ccc gtc     2304 Met Ser Lys Glu Leu Val Pro Gly Ser Val Hi #s Leu Asn Thr Pro Val     730               #   735               #   740 gct gaa att gag cag tcg gca tcc ggc tgt ac #a gta cga tcg gcc tcg     2352 Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Th #r Val Arg Ser Ala Ser 745                 7 #50                 7 #55                 7 #60 ggc gcc gtg ttc cga agc aaa aag gtg gtg gt #t tcg tta ccg aca acc     2400 Gly Ala Val Phe Arg Ser Lys Lys Val Val Va #l Ser Leu Pro Thr Thr                 765   #               770   #               775 ttg tat ccc acc ttg aca ttt tca cca cct ct #t ccc gcc gag aag caa     2448 Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Le #u Pro Ala Glu Lys Gln             780       #           785       #           790 gca ttg gcg gaa aat tct atc ctg ggc tac ta #t agc aag ata gtc ttc     2496 Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Ty #r Ser Lys Ile Val Phe         795           #       800           #       805 gta tgg gac aag ccg tgg tgg cgc gaa caa gg #c ttc tcg ggc gtc ctc     2544 Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gl #y Phe Ser Gly Val Leu     810               #   815               #   820 caa tcg agc tgt gac ccc atc tca ttt gcc ag #a gat acc agc atc gac     2592 Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Ar #g Asp Thr Ser Ile Asp 825                 8 #30                 8 #35                 8 #40 gtc gat cga caa tgg tcc att acc tgt ttc at #g gtc gga gac ccg gga     2640 Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Me #t Val Gly Asp Pro Gly                 845   #               850   #               855 cgg aag tgg tcc caa cag tcc aag cag gta cg #a caa aag tct gtc tgg     2688 Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Ar #g Gln Lys Ser Val Trp             860       #           865       #           870 gac caa ctc cgc gca gcc tac gag aac gcc gg #g gcc caa gtc cca gag     2736 Asp Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gl #y Ala Gln Val Pro Glu         875           #       880           #       885 ccg gcc aac gtg ctc gaa atc gag tgg tcg aa #g cag cag tat ttc caa     2784 Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Ly #s Gln Gln Tyr Phe Gln     890               #   895               #   900 gga gct ccg agc gcc gtc tat ggg ctg aac ga #t ctc atc aca ctg ggt     2832 Gly Ala Pro Ser Ala Val Tyr Gly Leu Asn As #p Leu Ile Thr Leu Gly 905                 9 #10                 9 #15                 9 #20 tcg gcg ctc aga acg ccg ttc aag agt gtt ca #t ttc gtt gga acg gag     2880 Ser Ala Leu Arg Thr Pro Phe Lys Ser Val Hi #s Phe Val Gly Thr Glu                 925   #               930   #               935 acg tct tta gtt tgg aaa ggg tat atg gaa gg #g gcc ata cga tcg ggt     2928 Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gl #y Ala Ile Arg Ser Gly             940       #           945       #           950 caa cga ggt gct gca gaa gtt gtg gct agc ct #g gtg cca gca gca         2973 Gln Arg Gly Ala Ala Glu Val Val Ala Ser Le #u Val Pro Ala Ala         955           #       960           #       965 tag                   #                   #                   #           2976 <210> SEQ ID NO 27 <211> LENGTH: 991 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: SIGNAL <222> LOCATION: (1)...(24) <400> SEQUENCE: 27 Met Ala Asn Lys His Leu Ser Leu Ser Leu Ph #e Leu Val Leu Leu Gly                 -20   #               -15   #               -10 Leu Ser Ala Ser Leu Ala Ser Gly Thr Asp Ph #e Pro Val Arg Arg Thr             -5       #             1      #          5 Asp Leu Gly Gln Val Gln Gly Leu Ala Gly As #p Val Met Ser Phe Arg     10               #    15               #    20 Gly Ile Pro Tyr Ala Ala Pro Pro Val Gly Gl #y Leu Arg Trp Lys Pro 25                   #30                   #35                   #40 Pro Gln His Ala Arg Pro Trp Ala Gly Val Ar #g Pro Ala Thr Gln Phe                 45   #                50   #                55 Gly Ser Asp Cys Phe Gly Ala Ala Tyr Leu Ar #g Lys Gly Ser Leu Ala             60       #            65       #            70 Pro Gly Val Ser Glu Asp Cys Leu Tyr Leu As #n Val Trp Ala Pro Ser         75           #        80           #        85 Gly Ala Lys Pro Gly Gln Tyr Pro Val Met Va #l Trp Val Tyr Gly Gly     90               #    95               #    100 Gly Phe Ala Gly Gly Thr Ala Ala Met Pro Ty #r Tyr Asp Gly Glu Ala 105                 1 #10                 1 #15                 1 #20 Leu Ala Arg Gln Gly Val Val Val Val Thr Ph #e Asn Tyr Arg Thr Asn                 125   #               130   #               135 Ile Leu Gly Phe Phe Ala His Pro Gly Leu Se #r Arg Glu Ser Pro Thr             140       #           145       #           150 Gly Thr Ser Gly Asn Tyr Gly Leu Leu Asp Il #e Leu Ala Ala Leu Arg         155           #       160           #       165 Trp Val Gln Ser Asn Ala Arg Ala Phe Gly Gl #y Asp Pro Gly Arg Val     170               #   175               #   180 Thr Val Phe Gly Glu Ser Ala Gly Ala Ser Al #a Ile Gly Leu Leu Leu 185                 1 #90                 1 #95                 2 #00 Thr Ser Pro Leu Ser Lys Gly Leu Phe Arg Gl #y Ala Ile Leu Glu Ser                 205   #               210   #               215 Pro Gly Leu Thr Arg Pro Leu Ala Thr Leu Al #a Asp Ser Ala Ala Ser             220       #           225       #           230 Gly Glu Arg Leu Asp Ala Asp Leu Ser Arg Le #u Arg Ser Thr Asp Pro         235           #       240           #       245 Ala Thr Leu Met Ala Arg Ala Asp Ala Ala Ar #g Pro Ala Ser Arg Asp     250               #   255               #   260 Leu Arg Arg Pro Arg Pro Thr Gly Pro Ile Va #l Asp Gly His Val Leu 265                 2 #70                 2 #75                 2 #80 Pro Gln Thr Asp Ser Ala Ala Ile Ala Ala Gl #y Gln Leu Ala Pro Val                 285   #               290   #               295 Arg Val Leu Ile Gly Thr Asn Ala Asp Glu Gl #y Arg Ala Phe Leu Gly             300       #           305       #           310 Arg Ala Pro Met Glu Thr Pro Ala Asp Tyr Gl #n Ala Tyr Leu Glu Ala         315           #       320           #       325 Gln Phe Gly Asp Gln Ala Ala Ala Val Ala Al #a Cys Tyr Pro Leu Asp     330               #   335               #   340 Gly Arg Ala Thr Pro Lys Glu Met Val Ala Ar #g Ile Phe Gly Asp Asn 345                 3 #50                 3 #55                 3 #60 Gln Phe Asn Arg Gly Val Ser Ala Phe Ser Gl #u Ala Leu Val Arg Gln                 365   #               370   #               375 Gly Ala Pro Val Trp Arg Tyr Gln Phe Asn Gl #y Asn Thr Glu Gly Gly             380       #           385       #           390 Arg Ala Pro Ala Thr His Gly Ala Glu Ile Pr #o Tyr Val Phe Gly Val         395           #       400           #       405 Phe Lys Leu Asp Glu Leu Gly Leu Phe Asp Tr #p Pro Pro Glu Gly Pro     410               #   415               #   420 Thr Pro Ala Asp Arg Ala Leu Gly Gln Leu Me #t Ser Ser Ala Trp Val 425                 4 #30                 4 #35                 4 #40 Arg Phe Ala Lys Asn Gly Asp Pro Ala Gly As #p Ala Leu Thr Trp Pro                 445   #               450   #               455 Ala Tyr Ser Thr Gly Lys Ser Thr Met Thr Ph #e Gly Pro Glu Gly Arg             460       #           465       #           470 Ala Ala Val Val Ser Pro Gly Pro Ser Ile Pr #o Pro Cys Ala Asp Gly         475           #       480           #       485 Ala Lys Ala Gly Gly Gly Gly Ser Gly Gly Gl #y Ser Gly Gly Gly Ser     490               #   495               #   500 Lys Asp Asn Val Ala Asp Val Val Val Val Gl #y Ala Gly Leu Ser Gly 505                 5 #10                 5 #15                 5 #20 Leu Glu Thr Ala Arg Lys Val Gln Ala Ala Gl #y Leu Ser Cys Leu Val                 525   #               530   #               535 Leu Glu Ala Met Asp Arg Val Gly Gly Lys Th #r Leu Ser Val Gln Ser             540       #           545       #           550 Gly Pro Gly Arg Thr Thr Ile Asn Asp Leu Gl #y Ala Ala Trp Ile Asn         555           #       560           #       565 Asp Ser Asn Gln Ser Glu Val Ser Arg Leu Ph #e Glu Arg Phe His Leu     570               #   575               #   580 Glu Gly Glu Leu Gln Arg Thr Thr Gly Asn Se #r Ile His Gln Ala Gln 585                 5 #90                 5 #95                 6 #00 Asp Gly Thr Thr Thr Thr Ala Pro Tyr Gly As #p Ser Leu Leu Ser Glu                 605   #               610   #               615 Glu Val Ala Ser Ala Leu Ala Glu Leu Leu Pr #o Val Trp Ser Gln Leu             620       #           625       #           630 Ile Glu Glu His Ser Leu Gln Asp Leu Lys Al #a Ser Pro Gln Ala Lys         635           #       640           #       645 Arg Leu Asp Ser Val Ser Phe Ala His Tyr Cy #s Glu Lys Glu Leu Asn     650               #   655               #   660 Leu Pro Ala Val Leu Gly Val Ala Asn Gln Il #e Thr Arg Ala Leu Leu 665                 6 #70                 6 #75                 6 #80 Gly Val Glu Ala His Glu Ile Ser Met Leu Ph #e Leu Thr Asp Tyr Ile                 685   #               690   #               695 Lys Ser Ala Thr Gly Leu Ser Asn Ile Phe Se #r Asp Lys Lys Asp Gly             700       #           705       #           710 Gly Gln Tyr Met Arg Cys Lys Thr Gly Met Gl #n Ser Ile Cys His Ala         715           #       720           #       725 Met Ser Lys Glu Leu Val Pro Gly Ser Val Hi #s Leu Asn Thr Pro Val     730               #   735               #   740 Ala Glu Ile Glu Gln Ser Ala Ser Gly Cys Th #r Val Arg Ser Ala Ser 745                 7 #50                 7 #55                 7 #60 Gly Ala Val Phe Arg Ser Lys Lys Val Val Va #l Ser Leu Pro Thr Thr                 765   #               770   #               775 Leu Tyr Pro Thr Leu Thr Phe Ser Pro Pro Le #u Pro Ala Glu Lys Gln             780       #           785       #           790 Ala Leu Ala Glu Asn Ser Ile Leu Gly Tyr Ty #r Ser Lys Ile Val Phe         795           #       800           #       805 Val Trp Asp Lys Pro Trp Trp Arg Glu Gln Gl #y Phe Ser Gly Val Leu     810               #   815               #   820 Gln Ser Ser Cys Asp Pro Ile Ser Phe Ala Ar #g Asp Thr Ser Ile Asp 825                 8 #30                 8 #35                 8 #40 Val Asp Arg Gln Trp Ser Ile Thr Cys Phe Me #t Val Gly Asp Pro Gly                 845   #               850   #               855 Arg Lys Trp Ser Gln Gln Ser Lys Gln Val Ar #g Gln Lys Ser Val Trp             860       #           865       #           870 Asp Gln Leu Arg Ala Ala Tyr Glu Asn Ala Gl #y Ala Gln Val Pro Glu         875           #       880           #       885 Pro Ala Asn Val Leu Glu Ile Glu Trp Ser Ly #s Gln Gln Tyr Phe Gln     890               #   895               #   900 Gly Ala Pro Ser Ala Val Tyr Gly Leu Asn As #p Leu Ile Thr Leu Gly 905                 9 #10                 9 #15                 9 #20 Ser Ala Leu Arg Thr Pro Phe Lys Ser Val Hi #s Phe Val Gly Thr Glu                 925   #               930   #               935 Thr Ser Leu Val Trp Lys Gly Tyr Met Glu Gl #y Ala Ile Arg Ser Gly             940       #           945       #           950 Gln Arg Gly Ala Ala Glu Val Val Ala Ser Le #u Val Pro Ala Ala         955           #       960           #       965 <210> SEQ ID NO 28 <211> LENGTH: 3618 <212> TYPE: DNA <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: gst:esp1:sp:K:trapao, 3618.  #1-687, gst +       polylinker; 688-2190, esp1 mat; 2191 #-2226 spacer; 2227-3615,       K:trAPAO, extra lysine; 3616-3618, s #top codon. For bacterial       expression. <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(3615) <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(687) <223> OTHER INFORMATION: gast + polylinker <220> FEATURE: <221> NAME/KEY: mat_peptide <222> LOCATION: (688)...(2190) <223> OTHER INFORMATION: esp1 mat <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (2191)...(2226) <223> OTHER INFORMATION: spacer sequence <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (2227)...(3615) <223> OTHER INFORMATION: K:trAPAO <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (2227)...(2229) <223> OTHER INFORMATION: Extra lysine <400> SEQUENCE: 28 atg tcc cct ata cta ggt tat tgg aaa att aa #g ggc ctt gtg caa ccc       48 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Ly #s Gly Leu Val Gln Pro  1               5   #                 10  #                 15 act cga ctt ctt ttg gaa tat ctt gaa gaa aa #a tat gaa gag cat ttg       96 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Ly #s Tyr Glu Glu His Leu              20      #             25      #             30 tat gag cgc gat gaa ggt gat aaa tgg cga aa #c aaa aag ttt gaa ttg      144 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg As #n Lys Lys Phe Glu Leu          35          #         40          #         45 ggt ttg gag ttt ccc aat ctt cct tat tat at #t gat ggt gat gtt aaa      192 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Il #e Asp Gly Asp Val Lys      50              #     55              #     60 tta aca cag tct atg gcc atc ata cgt tat at #a gct gac aag cac aac      240 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Il #e Ala Asp Lys His Asn  65                  # 70                  # 75                  # 80 atg ttg ggt ggt tgt cca aaa gag cgt gca ga #g att tca atg ctt gaa      288 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Gl #u Ile Ser Met Leu Glu                  85  #                 90  #                 95 gga gcg gtt ttg gat att aga tac ggt gtt tc #g aga att gca tat agt      336 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Se #r Arg Ile Ala Tyr Ser             100       #           105       #           110 aaa gac ttt gaa act ctc aaa gtt gat ttt ct #t agc aag cta cct gaa      384 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Le #u Ser Lys Leu Pro Glu         115           #       120           #       125 atg ctg aaa atg ttc gaa gat cgt tta tgt ca #t aaa aca tat tta aat      432 Met Leu Lys Met Phe Glu Asp Arg Leu Cys Hi #s Lys Thr Tyr Leu Asn     130               #   135               #   140 ggt gat cat gta acc cat cct gac ttc atg tt #g tat gac gct ctt gat      480 Gly Asp His Val Thr His Pro Asp Phe Met Le #u Tyr Asp Ala Leu Asp 145                 1 #50                 1 #55                 1 #60 gtt gtt tta tac atg gac cca atg tgc ctg ga #t gcg ttc cca aaa tta      528 Val Val Leu Tyr Met Asp Pro Met Cys Leu As #p Ala Phe Pro Lys Leu                 165   #               170   #               175 gtt tgt ttt aaa aaa cgt att gaa gct atc cc #a caa att gat aag tac      576 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pr #o Gln Ile Asp Lys Tyr             180       #           185       #           190 ttg aaa tcc agc aag tat ata gca tgg cct tt #g cag ggc tgg caa gcc      624 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Le #u Gln Gly Trp Gln Ala         195           #       200           #       205 acg ttt ggt ggt ggc gac cat cct cca aaa tc #g gat ctg gtt ccg cgt      672 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Se #r Asp Leu Val Pro Arg     210               #   215               #   220 gga tcc ccg gaa ttc gct cct act gtc aag at #t gat gct ggg atg gtg      720 Gly Ser Pro Glu Phe Ala Pro Thr Val Lys Il #e Asp Ala Gly Met Val 225                 2 #30                 2 #35                 2 #40 gtc ggc acg act act act gtc ccc ggc acc ac #t gcg acc gtc agc gag      768 Val Gly Thr Thr Thr Thr Val Pro Gly Thr Th #r Ala Thr Val Ser Glu                 245   #               250   #               255 ttc ttg ggc gtt cct ttt gcc gcc tct ccg ac #a cga ttt gcg cct cct      816 Phe Leu Gly Val Pro Phe Ala Ala Ser Pro Th #r Arg Phe Ala Pro Pro             260       #           265       #           270 act cgt ccc gtg cct tgg tca acg cct ttg ca #a gcc act gca tat ggt      864 Thr Arg Pro Val Pro Trp Ser Thr Pro Leu Gl #n Ala Thr Ala Tyr Gly         275           #       280           #       285 cca gca tgc cct caa caa ttc aat tac ccc ga #a gaa ctc cgt gag att      912 Pro Ala Cys Pro Gln Gln Phe Asn Tyr Pro Gl #u Glu Leu Arg Glu Ile     290               #   295               #   300 acg atg gcc tgg ttc aat aca ccg ccc ccg tc #a gct ggt gaa agt gag      960 Thr Met Ala Trp Phe Asn Thr Pro Pro Pro Se #r Ala Gly Glu Ser Glu 305                 3 #10                 3 #15                 3 #20 gac tgc ctg aac ctc aac atc tac gtc cca gg #a act gag aac aca aac     1008 Asp Cys Leu Asn Leu Asn Ile Tyr Val Pro Gl #y Thr Glu Asn Thr Asn                 325   #               330   #               335 aaa gcc gtc atg gtt tgg ata tac ggt gga gc #g ctg gaa tat ggt tgg     1056 Lys Ala Val Met Val Trp Ile Tyr Gly Gly Al #a Leu Glu Tyr Gly Trp             340       #           345       #           350 aat tca ttc cac ctt tac gac ggg gct agt tt #c gca gcc aat cag gat     1104 Asn Ser Phe His Leu Tyr Asp Gly Ala Ser Ph #e Ala Ala Asn Gln Asp         355           #       360           #       365 gtc atc gcc gtg acc atc aac tac aga acg aa #c att ctg ggg ttc cct     1152 Val Ile Ala Val Thr Ile Asn Tyr Arg Thr As #n Ile Leu Gly Phe Pro     370               #   375               #   380 gct gcc cct cag ctt cca ata aca cag cga aa #t ctg ggg ttc cta gac     1200 Ala Ala Pro Gln Leu Pro Ile Thr Gln Arg As #n Leu Gly Phe Leu Asp 385                 3 #90                 3 #95                 4 #00 caa agg ttt gct ttg gat tgg gta cag cgg aa #c atc gca gcc ttt ggc     1248 Gln Arg Phe Ala Leu Asp Trp Val Gln Arg As #n Ile Ala Ala Phe Gly                 405   #               410   #               415 ggt gat cct cga aag gtc aca ata ttt ggg ca #g agt gcg ggg ggc aga     1296 Gly Asp Pro Arg Lys Val Thr Ile Phe Gly Gl #n Ser Ala Gly Gly Arg             420       #           425       #           430 agt gtc gac gtc ctc ttg acg tct atg cca ca #c aac cca ccc ttc cga     1344 Ser Val Asp Val Leu Leu Thr Ser Met Pro Hi #s Asn Pro Pro Phe Arg         435           #       440           #       445 gca gca atc atg gag tcc ggt gtg gct aac ta #c aac ttc ccc aag gga     1392 Ala Ala Ile Met Glu Ser Gly Val Ala Asn Ty #r Asn Phe Pro Lys Gly     450               #   455               #   460 gat ttg tcc gaa cct tgg aac acc act gtt ca #a gct ctc aac tgt acc     1440 Asp Leu Ser Glu Pro Trp Asn Thr Thr Val Gl #n Ala Leu Asn Cys Thr 465                 4 #70                 4 #75                 4 #80 acc agt atc gac atc ttg agt tgt atg aga ag #a gtc gat ctc gcc act     1488 Thr Ser Ile Asp Ile Leu Ser Cys Met Arg Ar #g Val Asp Leu Ala Thr                 485   #               490   #               495 ctg atg aac acg atc gag caa ctc gga ctt gg #g ttt gag tac acg ttg     1536 Leu Met Asn Thr Ile Glu Gln Leu Gly Leu Gl #y Phe Glu Tyr Thr Leu             500       #           505       #           510 gac aac gta acg gct gtg tac cgt tct gaa ac #g gct cgc acg act ggt     1584 Asp Asn Val Thr Ala Val Tyr Arg Ser Glu Th #r Ala Arg Thr Thr Gly         515           #       520           #       525 gac att gct cgt gta cct gtt ctc gtc ggg ac #g gtg gcc aac gac gga     1632 Asp Ile Ala Arg Val Pro Val Leu Val Gly Th #r Val Ala Asn Asp Gly     530               #   535               #   540 ctt ctc ttt gtc ctc ggg gag aat gac acc ca #a gca tat ctc gag gag     1680 Leu Leu Phe Val Leu Gly Glu Asn Asp Thr Gl #n Ala Tyr Leu Glu Glu 545                 5 #50                 5 #55                 5 #60 gca atc ccg aat cag ccc gac ctt tac cag ac #t ctc ctt gga gca tat     1728 Ala Ile Pro Asn Gln Pro Asp Leu Tyr Gln Th #r Leu Leu Gly Ala Tyr                 565   #               570   #               575 ccc att gga tcc cca ggg atc gga tcg cct ca #a gat cag att gcc gcc     1776 Pro Ile Gly Ser Pro Gly Ile Gly Ser Pro Gl #n Asp Gln Ile Ala Ala             580       #           585       #           590 att gag acc gag gta aga ttc cag tgt cct tc #t gcc atc gtg gct cag     1824 Ile Glu Thr Glu Val Arg Phe Gln Cys Pro Se #r Ala Ile Val Ala Gln         595           #       600           #       605 gac tcc cgg aat cgg ggt atc cct tct tgg cg #c tac tac tac aat gcg     1872 Asp Ser Arg Asn Arg Gly Ile Pro Ser Trp Ar #g Tyr Tyr Tyr Asn Ala     610               #   615               #   620 acc ttt gag aat ctg gag ctt ttc cct ggg tc #c gaa gtg tac cac agc     1920 Thr Phe Glu Asn Leu Glu Leu Phe Pro Gly Se #r Glu Val Tyr His Ser 625                 6 #30                 6 #35                 6 #40 tct gaa gtc ggg atg gtg ttt ggc acg tat cc #t gtc gca agt gcg acc     1968 Ser Glu Val Gly Met Val Phe Gly Thr Tyr Pr #o Val Ala Ser Ala Thr                 645   #               650   #               655 gcc ttg gag gcc cag acg agc aaa tac atg ca #g ggt gcc tgg gcg gcc     2016 Ala Leu Glu Ala Gln Thr Ser Lys Tyr Met Gl #n Gly Ala Trp Ala Ala             660       #           665       #           670 ttt gcc aaa aac ccc atg aat ggg cct ggg tg #g aaa caa gtg ccg aat     2064 Phe Ala Lys Asn Pro Met Asn Gly Pro Gly Tr #p Lys Gln Val Pro Asn         675           #       680           #       685 gtc gcg gcg ctt ggc tca cca ggc aaa gcc at #c cag gtt gac gtc tct     2112 Val Ala Ala Leu Gly Ser Pro Gly Lys Ala Il #e Gln Val Asp Val Ser     690               #   695               #   700 cca gcg aca ata gac caa cga tgt gcc ttg ta #c acg cgt tat tat act     2160 Pro Ala Thr Ile Asp Gln Arg Cys Ala Leu Ty #r Thr Arg Tyr Tyr Thr 705                 7 #10                 7 #15                 7 #20 gag ttg ggc aca atc gcg ccg agg aca ttt gg #c gga ggc agc ggc gga     2208 Glu Leu Gly Thr Ile Ala Pro Arg Thr Phe Gl #y Gly Gly Ser Gly Gly                 725   #               730   #               735 ggc agc ggc gga ggc agc aaa gac aac gtt gc #g gac gtg gta gtg gtg     2256 Gly Ser Gly Gly Gly Ser Lys Asp Asn Val Al #a Asp Val Val Val Val             740       #           745       #           750 ggc gct ggc ttg agc ggt ttg gag acg gca cg #c aaa gtc cag gcc gcc     2304 Gly Ala Gly Leu Ser Gly Leu Glu Thr Ala Ar #g Lys Val Gln Ala Ala         755           #       760           #       765 ggt ctg tcc tgc ctc gtt ctt gag gcg atg ga #t cgt gta ggg gga aag     2352 Gly Leu Ser Cys Leu Val Leu Glu Ala Met As #p Arg Val Gly Gly Lys     770               #   775               #   780 act ctg agc gta caa tcg ggt ccc ggc agg ac #g act atc aac gac ctc     2400 Thr Leu Ser Val Gln Ser Gly Pro Gly Arg Th #r Thr Ile Asn Asp Leu 785                 7 #90                 7 #95                 8 #00 ggc gct gcg tgg atc aat gac agc aac caa ag #c gaa gta tcc aga ttg     2448 Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Se #r Glu Val Ser Arg Leu                 805   #               810   #               815 ttt gaa aga ttt cat ttg gag ggc gag ctc ca #g agg acg act gga aat     2496 Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gl #n Arg Thr Thr Gly Asn             820       #           825       #           830 tca atc cat caa gca caa gac ggt aca acc ac #t aca gct cct tat ggt     2544 Ser Ile His Gln Ala Gln Asp Gly Thr Thr Th #r Thr Ala Pro Tyr Gly         835           #       840           #       845 gac tcc ttg ctg agc gag gag gtt gca agt gc #a ctt gcg gaa ctc ctc     2592 Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Al #a Leu Ala Glu Leu Leu     850               #   855               #   860 ccc gta tgg tct cag ctg atc gaa gag cat ag #c ctt caa gac ctc aag     2640 Pro Val Trp Ser Gln Leu Ile Glu Glu His Se #r Leu Gln Asp Leu Lys 865                 8 #70                 8 #75                 8 #80 gcg agc cct cag gcg aag cgg ctc gac agt gt #g agc ttc gcg cac tac     2688 Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Va #l Ser Phe Ala His Tyr                 885   #               890   #               895 tgt gag aag gaa cta aac ttg cct gct gtt ct #c ggc gta gca aac cag     2736 Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Le #u Gly Val Ala Asn Gln             900       #           905       #           910 atc aca cgc gct ctg ctc ggt gtg gaa gcc ca #c gag atc agc atg ctt     2784 Ile Thr Arg Ala Leu Leu Gly Val Glu Ala Hi #s Glu Ile Ser Met Leu         915           #       920           #       925 ttt ctc acc gac tac atc aag agt gcc acc gg #t ctc agt aat att ttc     2832 Phe Leu Thr Asp Tyr Ile Lys Ser Ala Thr Gl #y Leu Ser Asn Ile Phe     930               #   935               #   940 tcg gac aag aaa gac ggc ggg cag tat atg cg #a tgc aaa aca ggt atg     2880 Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Ar #g Cys Lys Thr Gly Met 945                 9 #50                 9 #55                 9 #60 cag tcg att tgc cat gcc atg tca aag gaa ct #t gtt cca ggc tca gtg     2928 Gln Ser Ile Cys His Ala Met Ser Lys Glu Le #u Val Pro Gly Ser Val                 965   #               970   #               975 cac ctc aac acc ccc gtc gct gaa att gag ca #g tcg gca tcc ggc tgt     2976 His Leu Asn Thr Pro Val Ala Glu Ile Glu Gl #n Ser Ala Ser Gly Cys             980       #           985       #           990 aca gta cga tcg gcc tcg ggc gcc gtg ttc cg #a agc aaa aag gtg gtg     3024 Thr Val Arg Ser Ala Ser Gly Ala Val Phe Ar #g Ser Lys Lys Val Val          995          #       1000           #      1005 gtt tcg tta ccg aca acc ttg tat ccc acc tt #g aca ttt tca cca cct     3072 Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Le #u Thr Phe Ser Pro Pro     1010              #   1015               #  1020 ctt ccc gcc gag aag caa gca ttg gcg gaa aa #t tct atc ctg ggc tac     3120 Leu Pro Ala Glu Lys Gln Ala Leu Ala Glu As #n Ser Ile Leu Gly Tyr 1025                1030 #                1035  #               1040 tat agc aag ata gtc ttc gta tgg gac aag cc #g tgg tgg cgc gaa caa     3168 Tyr Ser Lys Ile Val Phe Val Trp Asp Lys Pr #o Trp Trp Arg Glu Gln                 1045  #               1050   #              1055 ggc ttc tcg ggc gtc ctc caa tcg agc tgt ga #c ccc atc tca ttt gcc     3216 Gly Phe Ser Gly Val Leu Gln Ser Ser Cys As #p Pro Ile Ser Phe Ala             1060      #           1065       #          1070 aga gat acc agc atc gac gtc gat cga caa tg #g tcc att acc tgt ttc     3264 Arg Asp Thr Ser Ile Asp Val Asp Arg Gln Tr #p Ser Ile Thr Cys Phe         1075          #       1080           #      1085 atg gtc gga gac ccg gga cgg aag tgg tcc ca #a cag tcc aag cag gta     3312 Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gl #n Gln Ser Lys Gln Val     1090              #   1095               #  1100 cga caa aag tct gtc tgg gac caa ctc cgc gc #a gcc tac gag aac gcc     3360 Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Al #a Ala Tyr Glu Asn Ala 1105                1110 #                1115  #               1120 ggg gcc caa gtc cca gag ccg gcc aac gtg ct #c gaa atc gag tgg tcg     3408 Gly Ala Gln Val Pro Glu Pro Ala Asn Val Le #u Glu Ile Glu Trp Ser                 1125  #               1130   #              1135 aag cag cag tat ttc caa gga gct ccg agc gc #c gtc tat ggg ctg aac     3456 Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Al #a Val Tyr Gly Leu Asn             1140      #           1145       #          1150 gat ctc atc aca ctg ggt tcg gcg ctc aga ac #g ccg ttc aag agt gtt     3504 Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Th #r Pro Phe Lys Ser Val         1155          #       1160           #      1165 cat ttc gtt gga acg gag acg tct tta gtt tg #g aaa ggg tat atg gaa     3552 His Phe Val Gly Thr Glu Thr Ser Leu Val Tr #p Lys Gly Tyr Met Glu     1170              #   1175               #  1180 ggg gcc ata cga tcg ggt caa cga ggt gct gc #a gaa gtt gtg gct agc     3600 Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Al #a Glu Val Val Ala Ser 1185                1190 #                1195  #               1200 ctg gtg cca gca gca tag          #                   #                   #3618 Leu Val Pro Ala Ala                 1205 <210> SEQ ID NO 29 <211> LENGTH: 1205 <212> TYPE: PRT <213> ORGANISM: Unknown <400> SEQUENCE: 29 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Ly #s Gly Leu Val Gln Pro  1               5   #                10   #                15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Ly #s Tyr Glu Glu His Leu             20       #            25       #            30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg As #n Lys Lys Phe Glu Leu         35           #        40           #        45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Il #e Asp Gly Asp Val Lys     50               #    55               #    60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Il #e Ala Asp Lys His Asn 65                   #70                   #75                   #80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Gl #u Ile Ser Met Leu Glu                 85   #                90   #                95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Se #r Arg Ile Ala Tyr Ser             100       #           105       #           110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Le #u Ser Lys Leu Pro Glu         115           #       120           #       125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys Hi #s Lys Thr Tyr Leu Asn     130               #   135               #   140 Gly Asp His Val Thr His Pro Asp Phe Met Le #u Tyr Asp Ala Leu Asp 145                 1 #50                 1 #55                 1 #60 Val Val Leu Tyr Met Asp Pro Met Cys Leu As #p Ala Phe Pro Lys Leu                 165   #               170   #               175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pr #o Gln Ile Asp Lys Tyr             180       #           185       #           190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Le #u Gln Gly Trp Gln Ala         195           #       200           #       205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Se #r Asp Leu Val Pro Arg     210               #   215               #   220 Gly Ser Pro Glu Phe Ala Pro Thr Val Lys Il #e Asp Ala Gly Met Val 225                 2 #30                 2 #35                 2 #40 Val Gly Thr Thr Thr Thr Val Pro Gly Thr Th #r Ala Thr Val Ser Glu                 245   #               250   #               255 Phe Leu Gly Val Pro Phe Ala Ala Ser Pro Th #r Arg Phe Ala Pro Pro             260       #           265       #           270 Thr Arg Pro Val Pro Trp Ser Thr Pro Leu Gl #n Ala Thr Ala Tyr Gly         275           #       280           #       285 Pro Ala Cys Pro Gln Gln Phe Asn Tyr Pro Gl #u Glu Leu Arg Glu Ile     290               #   295               #   300 Thr Met Ala Trp Phe Asn Thr Pro Pro Pro Se #r Ala Gly Glu Ser Glu 305                 3 #10                 3 #15                 3 #20 Asp Cys Leu Asn Leu Asn Ile Tyr Val Pro Gl #y Thr Glu Asn Thr Asn                 325   #               330   #               335 Lys Ala Val Met Val Trp Ile Tyr Gly Gly Al #a Leu Glu Tyr Gly Trp             340       #           345       #           350 Asn Ser Phe His Leu Tyr Asp Gly Ala Ser Ph #e Ala Ala Asn Gln Asp         355           #       360           #       365 Val Ile Ala Val Thr Ile Asn Tyr Arg Thr As #n Ile Leu Gly Phe Pro     370               #   375               #   380 Ala Ala Pro Gln Leu Pro Ile Thr Gln Arg As #n Leu Gly Phe Leu Asp 385                 3 #90                 3 #95                 4 #00 Gln Arg Phe Ala Leu Asp Trp Val Gln Arg As #n Ile Ala Ala Phe Gly                 405   #               410   #               415 Gly Asp Pro Arg Lys Val Thr Ile Phe Gly Gl #n Ser Ala Gly Gly Arg             420       #           425       #           430 Ser Val Asp Val Leu Leu Thr Ser Met Pro Hi #s Asn Pro Pro Phe Arg         435           #       440           #       445 Ala Ala Ile Met Glu Ser Gly Val Ala Asn Ty #r Asn Phe Pro Lys Gly     450               #   455               #   460 Asp Leu Ser Glu Pro Trp Asn Thr Thr Val Gl #n Ala Leu Asn Cys Thr 465                 4 #70                 4 #75                 4 #80 Thr Ser Ile Asp Ile Leu Ser Cys Met Arg Ar #g Val Asp Leu Ala Thr                 485   #               490   #               495 Leu Met Asn Thr Ile Glu Gln Leu Gly Leu Gl #y Phe Glu Tyr Thr Leu             500       #           505       #           510 Asp Asn Val Thr Ala Val Tyr Arg Ser Glu Th #r Ala Arg Thr Thr Gly         515           #       520           #       525 Asp Ile Ala Arg Val Pro Val Leu Val Gly Th #r Val Ala Asn Asp Gly     530               #   535               #   540 Leu Leu Phe Val Leu Gly Glu Asn Asp Thr Gl #n Ala Tyr Leu Glu Glu 545                 5 #50                 5 #55                 5 #60 Ala Ile Pro Asn Gln Pro Asp Leu Tyr Gln Th #r Leu Leu Gly Ala Tyr                 565   #               570   #               575 Pro Ile Gly Ser Pro Gly Ile Gly Ser Pro Gl #n Asp Gln Ile Ala Ala             580       #           585       #           590 Ile Glu Thr Glu Val Arg Phe Gln Cys Pro Se #r Ala Ile Val Ala Gln         595           #       600           #       605 Asp Ser Arg Asn Arg Gly Ile Pro Ser Trp Ar #g Tyr Tyr Tyr Asn Ala     610               #   615               #   620 Thr Phe Glu Asn Leu Glu Leu Phe Pro Gly Se #r Glu Val Tyr His Ser 625                 6 #30                 6 #35                 6 #40 Ser Glu Val Gly Met Val Phe Gly Thr Tyr Pr #o Val Ala Ser Ala Thr                 645   #               650   #               655 Ala Leu Glu Ala Gln Thr Ser Lys Tyr Met Gl #n Gly Ala Trp Ala Ala             660       #           665       #           670 Phe Ala Lys Asn Pro Met Asn Gly Pro Gly Tr #p Lys Gln Val Pro Asn         675           #       680           #       685 Val Ala Ala Leu Gly Ser Pro Gly Lys Ala Il #e Gln Val Asp Val Ser     690               #   695               #   700 Pro Ala Thr Ile Asp Gln Arg Cys Ala Leu Ty #r Thr Arg Tyr Tyr Thr 705                 7 #10                 7 #15                 7 #20 Glu Leu Gly Thr Ile Ala Pro Arg Thr Phe Gl #y Gly Gly Ser Gly Gly                 725   #               730   #               735 Gly Ser Gly Gly Gly Ser Lys Asp Asn Val Al #a Asp Val Val Val Val             740       #           745       #           750 Gly Ala Gly Leu Ser Gly Leu Glu Thr Ala Ar #g Lys Val Gln Ala Ala         755           #       760           #       765 Gly Leu Ser Cys Leu Val Leu Glu Ala Met As #p Arg Val Gly Gly Lys     770               #   775               #   780 Thr Leu Ser Val Gln Ser Gly Pro Gly Arg Th #r Thr Ile Asn Asp Leu 785                 7 #90                 7 #95                 8 #00 Gly Ala Ala Trp Ile Asn Asp Ser Asn Gln Se #r Glu Val Ser Arg Leu                 805   #               810   #               815 Phe Glu Arg Phe His Leu Glu Gly Glu Leu Gl #n Arg Thr Thr Gly Asn             820       #           825       #           830 Ser Ile His Gln Ala Gln Asp Gly Thr Thr Th #r Thr Ala Pro Tyr Gly         835           #       840           #       845 Asp Ser Leu Leu Ser Glu Glu Val Ala Ser Al #a Leu Ala Glu Leu Leu     850               #   855               #   860 Pro Val Trp Ser Gln Leu Ile Glu Glu His Se #r Leu Gln Asp Leu Lys 865                 8 #70                 8 #75                 8 #80 Ala Ser Pro Gln Ala Lys Arg Leu Asp Ser Va #l Ser Phe Ala His Tyr                 885   #               890   #               895 Cys Glu Lys Glu Leu Asn Leu Pro Ala Val Le #u Gly Val Ala Asn Gln             900       #           905       #           910 Ile Thr Arg Ala Leu Leu Gly Val Glu Ala Hi #s Glu Ile Ser Met Leu         915           #       920           #       925 Phe Leu Thr Asp Tyr Ile Lys Ser Ala Thr Gl #y Leu Ser Asn Ile Phe     930               #   935               #   940 Ser Asp Lys Lys Asp Gly Gly Gln Tyr Met Ar #g Cys Lys Thr Gly Met 945                 9 #50                 9 #55                 9 #60 Gln Ser Ile Cys His Ala Met Ser Lys Glu Le #u Val Pro Gly Ser Val                 965   #               970   #               975 His Leu Asn Thr Pro Val Ala Glu Ile Glu Gl #n Ser Ala Ser Gly Cys             980       #           985       #           990 Thr Val Arg Ser Ala Ser Gly Ala Val Phe Ar #g Ser Lys Lys Val Val         995           #       1000           #      1005 Val Ser Leu Pro Thr Thr Leu Tyr Pro Thr Le #u Thr Phe Ser Pro Pro     1010              #   1015               #  1020 Leu Pro Ala Glu Lys Gln Ala Leu Ala Glu As #n Ser Ile Leu Gly Tyr 1025                1030 #                1035  #               1040 Tyr Ser Lys Ile Val Phe Val Trp Asp Lys Pr #o Trp Trp Arg Glu Gln                 1045  #               1050   #              1055 Gly Phe Ser Gly Val Leu Gln Ser Ser Cys As #p Pro Ile Ser Phe Ala             1060      #           1065       #          1070 Arg Asp Thr Ser Ile Asp Val Asp Arg Gln Tr #p Ser Ile Thr Cys Phe         1075          #       1080           #      1085 Met Val Gly Asp Pro Gly Arg Lys Trp Ser Gl #n Gln Ser Lys Gln Val     1090              #   1095               #  1100 Arg Gln Lys Ser Val Trp Asp Gln Leu Arg Al #a Ala Tyr Glu Asn Ala 1105                1110 #                1115  #               1120 Gly Ala Gln Val Pro Glu Pro Ala Asn Val Le #u Glu Ile Glu Trp Ser                 1125  #               1130   #              1135 Lys Gln Gln Tyr Phe Gln Gly Ala Pro Ser Al #a Val Tyr Gly Leu Asn             1140      #           1145       #          1150 Asp Leu Ile Thr Leu Gly Ser Ala Leu Arg Th #r Pro Phe Lys Ser Val         1155          #       1160           #      1165 His Phe Val Gly Thr Glu Thr Ser Leu Val Tr #p Lys Gly Tyr Met Glu     1170              #   1175               #  1180 Gly Ala Ile Arg Ser Gly Gln Arg Gly Ala Al #a Glu Val Val Ala Ser 1185                1190 #                1195  #               1200 Leu Val Pro Ala Ala                 1205 <210> SEQ ID NO 30 <211> LENGTH: 3591 <212> TYPE: DNA <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: Open reading frame of  #BEST1:K:trAPAO fusion for       bacterial expression vector pGEX-4T-1  #or similar vector.       gst:BEST1:sp:K:trAPAO fusion, 3591 nt.  #1-687 gst + polylinker,       688-2163, BEST1 mature; 2164-2199, s #pacer, 2200-3588, K:trAPAO <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(687) <223> OTHER INFORMATION: gst + polylinker <220> FEATURE: <221> NAME/KEY: mat_peptide <222> LOCATION: (688)...(2163) <223> OTHER INFORMATION: BEST1 mature <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (2164)...(2199) <223> OTHER INFORMATION: spacer sequence <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (2200)...(3588) <223> OTHER INFORMATION: K:trAPAO <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(3588) <221> NAME/KEY: misc_feature <222> LOCATION: (2200)...(2202) <223> OTHER INFORMATION: Extra lysine <400> SEQUENCE: 30 atg tcc cct ata cta ggt tat tgg aaa att aa #g ggc ctt gtg caa ccc       48 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Ly #s Gly Leu Val Gln Pro  1               5   #                 10  #                 15 act cga ctt ctt ttg gaa tat ctt gaa gaa aa #a tat gaa gag cat ttg       96 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Ly #s Tyr Glu Glu His Leu              20      #             25      #             30 tat gag cgc gat gaa ggt gat aaa tgg cga aa #c aaa aag ttt gaa ttg      144 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg As #n Lys Lys Phe Glu Leu          35          #         40          #         45 ggt ttg gag ttt ccc aat ctt cct tat tat at #t gat ggt gat gtt aaa      192 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Il #e Asp Gly Asp Val Lys      50              #     55              #     60 tta aca cag tct atg gcc atc ata cgt tat at #a gct gac aag cac aac      240 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Il #e Ala Asp Lys His Asn  65                  # 70                  # 75                  # 80 atg ttg ggt ggt tgt cca aaa gag cgt gca ga #g att tca atg ctt gaa      288 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Gl #u Ile Ser Met Leu Glu                  85  #                 90  #                 95 gga gcg gtt ttg gat att aga tac ggt gtt tc #g aga att gca tat agt      336 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Se #r Arg Ile Ala Tyr Ser             100       #           105       #           110 aaa gac ttt gaa act ctc aaa gtt gat ttt ct #t agc aag cta cct gaa      384 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Le #u Ser Lys Leu Pro Glu         115           #       120           #       125 atg ctg aaa atg ttc gaa gat cgt tta tgt ca #t aaa aca tat tta aat      432 Met Leu Lys Met Phe Glu Asp Arg Leu Cys Hi #s Lys Thr Tyr Leu Asn     130               #   135               #   140 ggt gat cat gta acc cat cct gac ttc atg tt #g tat gac gct ctt gat      480 Gly Asp His Val Thr His Pro Asp Phe Met Le #u Tyr Asp Ala Leu Asp 145                 1 #50                 1 #55                 1 #60 gtt gtt tta tac atg gac cca atg tgc ctg ga #t gcg ttc cca aaa tta      528 Val Val Leu Tyr Met Asp Pro Met Cys Leu As #p Ala Phe Pro Lys Leu                 165   #               170   #               175 gtt tgt ttt aaa aaa cgt att gaa gct atc cc #a caa att gat aag tac      576 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pr #o Gln Ile Asp Lys Tyr             180       #           185       #           190 ttg aaa tcc agc aag tat ata gca tgg cct tt #g cag ggc tgg caa gcc      624 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Le #u Gln Gly Trp Gln Ala         195           #       200           #       205 acg ttt ggt ggt ggc gac cat cct cca aaa tc #g gat ctg gtt ccg cgt      672 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Se #r Asp Leu Val Pro Arg     210               #   215               #   220 gga tcc ccg gaa ttc acg gat ttt ccg gtc cg #c agg acc gat ctg ggc      720 Gly Ser Pro Glu Phe Thr Asp Phe Pro Val Ar #g Arg Thr Asp Leu Gly 225                 2 #30                 2 #35                 2 #40 cag gtt cag gga ctg gcc ggg gac gtg atg ag #c ttt cgc gga ata ccc      768 Gln Val Gln Gly Leu Ala Gly Asp Val Met Se #r Phe Arg Gly Ile Pro                 245   #               250   #               255 tat gca gcg ccg ccg gtg ggc ggg ctg cgt tg #g aag ccg ccc caa cac      816 Tyr Ala Ala Pro Pro Val Gly Gly Leu Arg Tr #p Lys Pro Pro Gln His             260       #           265       #           270 gcc cgg ccc tgg gcg ggc gtt cgc ccc gcc ac #c caa ttt ggc tcc gac      864 Ala Arg Pro Trp Ala Gly Val Arg Pro Ala Th #r Gln Phe Gly Ser Asp         275           #       280           #       285 tgc ttc ggc gcg gcc tat ctt cgc aaa ggc ag #c ctc gcc ccc ggc gtg      912 Cys Phe Gly Ala Ala Tyr Leu Arg Lys Gly Se #r Leu Ala Pro Gly Val     290               #   295               #   300 agc gag gac tgt ctt tac ctc aac gta tgg gc #g ccg tca ggc gct aaa      960 Ser Glu Asp Cys Leu Tyr Leu Asn Val Trp Al #a Pro Ser Gly Ala Lys 305                 3 #10                 3 #15                 3 #20 ccc ggc cag tac ccc gtc atg gtc tgg gtc ta #c ggc ggc ggc ttc gcc     1008 Pro Gly Gln Tyr Pro Val Met Val Trp Val Ty #r Gly Gly Gly Phe Ala                 325   #               330   #               335 ggc ggc acg gcc gcc atg ccc tac tac gac gg #c gag gcg ctt gcg cga     1056 Gly Gly Thr Ala Ala Met Pro Tyr Tyr Asp Gl #y Glu Ala Leu Ala Arg             340       #           345       #           350 cag ggc gtc gtc gtg gtg acg ttt aac tat cg #g acg aac atc ctg ggc     1104 Gln Gly Val Val Val Val Thr Phe Asn Tyr Ar #g Thr Asn Ile Leu Gly         355           #       360           #       365 ttt ttc gcc cat cct ggt ctc tcg cgc gag ag #c ccc acc gga act tcg     1152 Phe Phe Ala His Pro Gly Leu Ser Arg Glu Se #r Pro Thr Gly Thr Ser     370               #   375               #   380 ggc aac tac ggc cta ctc gac att ctc gcc gc #t ctt cgg tgg gtg cag     1200 Gly Asn Tyr Gly Leu Leu Asp Ile Leu Ala Al #a Leu Arg Trp Val Gln 385                 3 #90                 3 #95                 4 #00 agc aac gcc cgc gcc ttc gga ggg gac ccc gg #c cga gtg acg gtc ttt     1248 Ser Asn Ala Arg Ala Phe Gly Gly Asp Pro Gl #y Arg Val Thr Val Phe                 405   #               410   #               415 ggt gaa tcg gcc gga gcg agc gcg atc gga ct #t ctg ctc acc tcg ccg     1296 Gly Glu Ser Ala Gly Ala Ser Ala Ile Gly Le #u Leu Leu Thr Ser Pro             420       #           425       #           430 ctg agc aag ggt ctc ttc cgt ggc gct atc ct #c gaa agt cca ggg ctg     1344 Leu Ser Lys Gly Leu Phe Arg Gly Ala Ile Le #u Glu Ser Pro Gly Leu         435           #       440           #       445 acg cga ccg ctc gcg acg ctc gcc gac agc gc #c gcc tcg ggc gag cgc     1392 Thr Arg Pro Leu Ala Thr Leu Ala Asp Ser Al #a Ala Ser Gly Glu Arg     450               #   455               #   460 ctc gac gcc gat ctt tcg cga ctg cgc tcg ac #c gac cca gcc acc ctg     1440 Leu Asp Ala Asp Leu Ser Arg Leu Arg Ser Th #r Asp Pro Ala Thr Leu 465                 4 #70                 4 #75                 4 #80 atg gcg cgc gcc gac gcg gcc cgc ccg gca tc #g cgg gac ctg cgc agg     1488 Met Ala Arg Ala Asp Ala Ala Arg Pro Ala Se #r Arg Asp Leu Arg Arg                 485   #               490   #               495 ccg cgt ccg acc gga ccg atc gtc gat ggc ca #t gtg ctg ccg cag acc     1536 Pro Arg Pro Thr Gly Pro Ile Val Asp Gly Hi #s Val Leu Pro Gln Thr             500       #           505       #           510 gac agc gcg gcg atc gcg gcg ggg cag ctg gc #g ccg gtt cgg gtc ctg     1584 Asp Ser Ala Ala Ile Ala Ala Gly Gln Leu Al #a Pro Val Arg Val Leu         515           #       520           #       525 atc gga acc aat gcc gac gaa ggc cgc gcc tt #c ctc ggg cgc gcg ccg     1632 Ile Gly Thr Asn Ala Asp Glu Gly Arg Ala Ph #e Leu Gly Arg Ala Pro     530               #   535               #   540 atg gag acg cca gcg gac tac caa gcc tat ct #g gag gcg cag ttt ggc     1680 Met Glu Thr Pro Ala Asp Tyr Gln Ala Tyr Le #u Glu Ala Gln Phe Gly 545                 5 #50                 5 #55                 5 #60 gac caa gcc gcc gcc gtg gcg gcg tgc tat cc #c ctc gac ggc cgg gcc     1728 Asp Gln Ala Ala Ala Val Ala Ala Cys Tyr Pr #o Leu Asp Gly Arg Ala                 565   #               570   #               575 acg ccc aag gaa atg gtc gcg cgc atc ttc gg #c gac aat cag ttc aat     1776 Thr Pro Lys Glu Met Val Ala Arg Ile Phe Gl #y Asp Asn Gln Phe Asn             580       #           585       #           590 cgg ggg gtc tcg gcc ttc tcg gaa gcg ctt gt #g cgc cag ggc gcg ccc     1824 Arg Gly Val Ser Ala Phe Ser Glu Ala Leu Va #l Arg Gln Gly Ala Pro         595           #       600           #       605 gtg tgg cgt tat cag ttc aac ggt aat acc ga #g ggt gga aga gcg ccg     1872 Val Trp Arg Tyr Gln Phe Asn Gly Asn Thr Gl #u Gly Gly Arg Ala Pro     610               #   615               #   620 gct acc cac gga gcc gaa att ccc tac gtt tt #c ggg gtg ttc aag ctc     1920 Ala Thr His Gly Ala Glu Ile Pro Tyr Val Ph #e Gly Val Phe Lys Leu 625                 6 #30                 6 #35                 6 #40 gac gag ttg ggt ctg ttc gat tgg ccg ccc ga #g ggg ccc acg ccc gcc     1968 Asp Glu Leu Gly Leu Phe Asp Trp Pro Pro Gl #u Gly Pro Thr Pro Ala                 645   #               650   #               655 gac cgt gcg ctg ggc caa ctg atg tcc tcc gc #c tgg gtc cgg ttc gcc     2016 Asp Arg Ala Leu Gly Gln Leu Met Ser Ser Al #a Trp Val Arg Phe Ala             660       #           665       #           670 aag aat ggc gac ccc gcc ggg gac gcc ctt ac #c tgg cct gcc tat tct     2064 Lys Asn Gly Asp Pro Ala Gly Asp Ala Leu Th #r Trp Pro Ala Tyr Ser         675           #       680           #       685 acg ggc aag tcg acc atg aca ttc ggt ccc ga #g ggc cgc gcg gcg gtg     2112 Thr Gly Lys Ser Thr Met Thr Phe Gly Pro Gl #u Gly Arg Ala Ala Val     690               #   695               #   700 gtg tcg ccc gga cct tcc atc ccc cct tgc gc #g gat ggc gcc aag gcg     2160 Val Ser Pro Gly Pro Ser Ile Pro Pro Cys Al #a Asp Gly Ala Lys Ala 705                 7 #10                 7 #15                 7 #20 ggg ggc gga ggc agc ggc gga ggc agc ggc gg #a ggc agc aaa gac aac     2208 Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gl #y Gly Ser Lys Asp Asn                 725   #               730   #               735 gtt gcg gac gtg gta gtg gtg ggc gct ggc tt #g agc ggt ttg gag acg     2256 Val Ala Asp Val Val Val Val Gly Ala Gly Le #u Ser Gly Leu Glu Thr             740       #           745       #           750 gca cgc aaa gtc cag gcc gcc ggt ctg tcc tg #c ctc gtt ctt gag gcg     2304 Ala Arg Lys Val Gln Ala Ala Gly Leu Ser Cy #s Leu Val Leu Glu Ala         755           #       760           #       765 atg gat cgt gta ggg gga aag act ctg agc gt #a caa tcg ggt ccc ggc     2352 Met Asp Arg Val Gly Gly Lys Thr Leu Ser Va #l Gln Ser Gly Pro Gly     770               #   775               #   780 agg acg act atc aac gac ctc ggc gct gcg tg #g atc aat gac agc aac     2400 Arg Thr Thr Ile Asn Asp Leu Gly Ala Ala Tr #p Ile Asn Asp Ser Asn 785                 7 #90                 7 #95                 8 #00 caa agc gaa gta tcc aga ttg ttt gaa aga tt #t cat ttg gag ggc gag     2448 Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Ph #e His Leu Glu Gly Glu                 805   #               810   #               815 ctc cag agg acg act gga aat tca atc cat ca #a gca caa gac ggt aca     2496 Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gl #n Ala Gln Asp Gly Thr             820       #           825       #           830 acc act aca gct cct tat ggt gac tcc ttg ct #g agc gag gag gtt gca     2544 Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Le #u Ser Glu Glu Val Ala         835           #       840           #       845 agt gca ctt gcg gaa ctc ctc ccc gta tgg tc #t cag ctg atc gaa gag     2592 Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Se #r Gln Leu Ile Glu Glu     850               #   855               #   860 cat agc ctt caa gac ctc aag gcg agc cct ca #g gcg aag cgg ctc gac     2640 His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gl #n Ala Lys Arg Leu Asp 865                 8 #70                 8 #75                 8 #80 agt gtg agc ttc gcg cac tac tgt gag aag ga #a cta aac ttg cct gct     2688 Ser Val Ser Phe Ala His Tyr Cys Glu Lys Gl #u Leu Asn Leu Pro Ala                 885   #               890   #               895 gtt ctc ggc gta gca aac cag atc aca cgc gc #t ctg ctc ggt gtg gaa     2736 Val Leu Gly Val Ala Asn Gln Ile Thr Arg Al #a Leu Leu Gly Val Glu             900       #           905       #           910 gcc cac gag atc agc atg ctt ttt ctc acc ga #c tac atc aag agt gcc     2784 Ala His Glu Ile Ser Met Leu Phe Leu Thr As #p Tyr Ile Lys Ser Ala         915           #       920           #       925 acc ggt ctc agt aat att ttc tcg gac aag aa #a gac ggc ggg cag tat     2832 Thr Gly Leu Ser Asn Ile Phe Ser Asp Lys Ly #s Asp Gly Gly Gln Tyr     930               #   935               #   940 atg cga tgc aaa aca ggt atg cag tcg att tg #c cat gcc atg tca aag     2880 Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cy #s His Ala Met Ser Lys 945                 9 #50                 9 #55                 9 #60 gaa ctt gtt cca ggc tca gtg cac ctc aac ac #c ccc gtc gct gaa att     2928 Glu Leu Val Pro Gly Ser Val His Leu Asn Th #r Pro Val Ala Glu Ile                 965   #               970   #               975 gag cag tcg gca tcc ggc tgt aca gta cga tc #g gcc tcg ggc gcc gtg     2976 Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Se #r Ala Ser Gly Ala Val             980       #           985       #           990 ttc cga agc aaa aag gtg gtg gtt tcg tta cc #g aca acc ttg tat ccc     3024 Phe Arg Ser Lys Lys Val Val Val Ser Leu Pr #o Thr Thr Leu Tyr Pro          995          #       1000           #      1005 acc ttg aca ttt tca cca cct ctt ccc gcc ga #g aag caa gca ttg gcg     3072 Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Gl #u Lys Gln Ala Leu Ala     1010              #   1015               #  1020 gaa aat tct atc ctg ggc tac tat agc aag at #a gtc ttc gta tgg gac     3120 Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Il #e Val Phe Val Trp Asp 1025                1030 #                1035  #               1040 aag ccg tgg tgg cgc gaa caa ggc ttc tcg gg #c gtc ctc caa tcg agc     3168 Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gl #y Val Leu Gln Ser Ser                 1045  #               1050   #              1055 tgt gac ccc atc tca ttt gcc aga gat acc ag #c atc gac gtc gat cga     3216 Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Se #r Ile Asp Val Asp Arg             1060      #           1065       #          1070 caa tgg tcc att acc tgt ttc atg gtc gga ga #c ccg gga cgg aag tgg     3264 Gln Trp Ser Ile Thr Cys Phe Met Val Gly As #p Pro Gly Arg Lys Trp         1075          #       1080           #      1085 tcc caa cag tcc aag cag gta cga caa aag tc #t gtc tgg gac caa ctc     3312 Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Se #r Val Trp Asp Gln Leu     1090              #   1095               #  1100 cgc gca gcc tac gag aac gcc ggg gcc caa gt #c cca gag ccg gcc aac     3360 Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gln Va #l Pro Glu Pro Ala Asn 1105                1110 #                1115  #               1120 gtg ctc gaa atc gag tgg tcg aag cag cag ta #t ttc caa gga gct ccg     3408 Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Ty #r Phe Gln Gly Ala Pro                 1125  #               1130   #              1135 agc gcc gtc tat ggg ctg aac gat ctc atc ac #a ctg ggt tcg gcg ctc     3456 Ser Ala Val Tyr Gly Leu Asn Asp Leu Ile Th #r Leu Gly Ser Ala Leu             1140      #           1145       #          1150 aga acg ccg ttc aag agt gtt cat ttc gtt gg #a acg gag acg tct tta     3504 Arg Thr Pro Phe Lys Ser Val His Phe Val Gl #y Thr Glu Thr Ser Leu         1155          #       1160           #      1165 gtt tgg aaa ggg tat atg gaa ggg gcc ata cg #a tcg ggt caa cga ggt     3552 Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Ar #g Ser Gly Gln Arg Gly     1170              #   1175               #  1180 gct gca gaa gtt gtg gct agc ctg gtg cca gc #a gca tag               #   3591 Ala Ala Glu Val Val Ala Ser Leu Val Pro Al #a Ala 1185                1190 #                1195 <210> SEQ ID NO 31 <211> LENGTH: 1196 <212> TYPE: PRT <213> ORGANISM: Unknown <400> SEQUENCE: 31 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Ly #s Gly Leu Val Gln Pro  1               5   #                10   #                15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Ly #s Tyr Glu Glu His Leu             20       #            25       #            30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg As #n Lys Lys Phe Glu Leu         35           #        40           #        45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Il #e Asp Gly Asp Val Lys     50               #    55               #    60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Il #e Ala Asp Lys His Asn 65                   #70                   #75                   #80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Gl #u Ile Ser Met Leu Glu                 85   #                90   #                95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Se #r Arg Ile Ala Tyr Ser             100       #           105       #           110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Le #u Ser Lys Leu Pro Glu         115           #       120           #       125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys Hi #s Lys Thr Tyr Leu Asn     130               #   135               #   140 Gly Asp His Val Thr His Pro Asp Phe Met Le #u Tyr Asp Ala Leu Asp 145                 1 #50                 1 #55                 1 #60 Val Val Leu Tyr Met Asp Pro Met Cys Leu As #p Ala Phe Pro Lys Leu                 165   #               170   #               175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pr #o Gln Ile Asp Lys Tyr             180       #           185       #           190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Le #u Gln Gly Trp Gln Ala         195           #       200           #       205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Se #r Asp Leu Val Pro Arg     210               #   215               #   220 Gly Ser Pro Glu Phe Thr Asp Phe Pro Val Ar #g Arg Thr Asp Leu Gly 225                 2 #30                 2 #35                 2 #40 Gln Val Gln Gly Leu Ala Gly Asp Val Met Se #r Phe Arg Gly Ile Pro                 245   #               250   #               255 Tyr Ala Ala Pro Pro Val Gly Gly Leu Arg Tr #p Lys Pro Pro Gln His             260       #           265       #           270 Ala Arg Pro Trp Ala Gly Val Arg Pro Ala Th #r Gln Phe Gly Ser Asp         275           #       280           #       285 Cys Phe Gly Ala Ala Tyr Leu Arg Lys Gly Se #r Leu Ala Pro Gly Val     290               #   295               #   300 Ser Glu Asp Cys Leu Tyr Leu Asn Val Trp Al #a Pro Ser Gly Ala Lys 305                 3 #10                 3 #15                 3 #20 Pro Gly Gln Tyr Pro Val Met Val Trp Val Ty #r Gly Gly Gly Phe Ala                 325   #               330   #               335 Gly Gly Thr Ala Ala Met Pro Tyr Tyr Asp Gl #y Glu Ala Leu Ala Arg             340       #           345       #           350 Gln Gly Val Val Val Val Thr Phe Asn Tyr Ar #g Thr Asn Ile Leu Gly         355           #       360           #       365 Phe Phe Ala His Pro Gly Leu Ser Arg Glu Se #r Pro Thr Gly Thr Ser     370               #   375               #   380 Gly Asn Tyr Gly Leu Leu Asp Ile Leu Ala Al #a Leu Arg Trp Val Gln 385                 3 #90                 3 #95                 4 #00 Ser Asn Ala Arg Ala Phe Gly Gly Asp Pro Gl #y Arg Val Thr Val Phe                 405   #               410   #               415 Gly Glu Ser Ala Gly Ala Ser Ala Ile Gly Le #u Leu Leu Thr Ser Pro             420       #           425       #           430 Leu Ser Lys Gly Leu Phe Arg Gly Ala Ile Le #u Glu Ser Pro Gly Leu         435           #       440           #       445 Thr Arg Pro Leu Ala Thr Leu Ala Asp Ser Al #a Ala Ser Gly Glu Arg     450               #   455               #   460 Leu Asp Ala Asp Leu Ser Arg Leu Arg Ser Th #r Asp Pro Ala Thr Leu 465                 4 #70                 4 #75                 4 #80 Met Ala Arg Ala Asp Ala Ala Arg Pro Ala Se #r Arg Asp Leu Arg Arg                 485   #               490   #               495 Pro Arg Pro Thr Gly Pro Ile Val Asp Gly Hi #s Val Leu Pro Gln Thr             500       #           505       #           510 Asp Ser Ala Ala Ile Ala Ala Gly Gln Leu Al #a Pro Val Arg Val Leu         515           #       520           #       525 Ile Gly Thr Asn Ala Asp Glu Gly Arg Ala Ph #e Leu Gly Arg Ala Pro     530               #   535               #   540 Met Glu Thr Pro Ala Asp Tyr Gln Ala Tyr Le #u Glu Ala Gln Phe Gly 545                 5 #50                 5 #55                 5 #60 Asp Gln Ala Ala Ala Val Ala Ala Cys Tyr Pr #o Leu Asp Gly Arg Ala                 565   #               570   #               575 Thr Pro Lys Glu Met Val Ala Arg Ile Phe Gl #y Asp Asn Gln Phe Asn             580       #           585       #           590 Arg Gly Val Ser Ala Phe Ser Glu Ala Leu Va #l Arg Gln Gly Ala Pro         595           #       600           #       605 Val Trp Arg Tyr Gln Phe Asn Gly Asn Thr Gl #u Gly Gly Arg Ala Pro     610               #   615               #   620 Ala Thr His Gly Ala Glu Ile Pro Tyr Val Ph #e Gly Val Phe Lys Leu 625                 6 #30                 6 #35                 6 #40 Asp Glu Leu Gly Leu Phe Asp Trp Pro Pro Gl #u Gly Pro Thr Pro Ala                 645   #               650   #               655 Asp Arg Ala Leu Gly Gln Leu Met Ser Ser Al #a Trp Val Arg Phe Ala             660       #           665       #           670 Lys Asn Gly Asp Pro Ala Gly Asp Ala Leu Th #r Trp Pro Ala Tyr Ser         675           #       680           #       685 Thr Gly Lys Ser Thr Met Thr Phe Gly Pro Gl #u Gly Arg Ala Ala Val     690               #   695               #   700 Val Ser Pro Gly Pro Ser Ile Pro Pro Cys Al #a Asp Gly Ala Lys Ala 705                 7 #10                 7 #15                 7 #20 Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gl #y Gly Ser Lys Asp Asn                 725   #               730   #               735 Val Ala Asp Val Val Val Val Gly Ala Gly Le #u Ser Gly Leu Glu Thr             740       #           745       #           750 Ala Arg Lys Val Gln Ala Ala Gly Leu Ser Cy #s Leu Val Leu Glu Ala         755           #       760           #       765 Met Asp Arg Val Gly Gly Lys Thr Leu Ser Va #l Gln Ser Gly Pro Gly     770               #   775               #   780 Arg Thr Thr Ile Asn Asp Leu Gly Ala Ala Tr #p Ile Asn Asp Ser Asn 785                 7 #90                 7 #95                 8 #00 Gln Ser Glu Val Ser Arg Leu Phe Glu Arg Ph #e His Leu Glu Gly Glu                 805   #               810   #               815 Leu Gln Arg Thr Thr Gly Asn Ser Ile His Gl #n Ala Gln Asp Gly Thr             820       #           825       #           830 Thr Thr Thr Ala Pro Tyr Gly Asp Ser Leu Le #u Ser Glu Glu Val Ala         835           #       840           #       845 Ser Ala Leu Ala Glu Leu Leu Pro Val Trp Se #r Gln Leu Ile Glu Glu     850               #   855               #   860 His Ser Leu Gln Asp Leu Lys Ala Ser Pro Gl #n Ala Lys Arg Leu Asp 865                 8 #70                 8 #75                 8 #80 Ser Val Ser Phe Ala His Tyr Cys Glu Lys Gl #u Leu Asn Leu Pro Ala                 885   #               890   #               895 Val Leu Gly Val Ala Asn Gln Ile Thr Arg Al #a Leu Leu Gly Val Glu             900       #           905       #           910 Ala His Glu Ile Ser Met Leu Phe Leu Thr As #p Tyr Ile Lys Ser Ala         915           #       920           #       925 Thr Gly Leu Ser Asn Ile Phe Ser Asp Lys Ly #s Asp Gly Gly Gln Tyr     930               #   935               #   940 Met Arg Cys Lys Thr Gly Met Gln Ser Ile Cy #s His Ala Met Ser Lys 945                 9 #50                 9 #55                 9 #60 Glu Leu Val Pro Gly Ser Val His Leu Asn Th #r Pro Val Ala Glu Ile                 965   #               970   #               975 Glu Gln Ser Ala Ser Gly Cys Thr Val Arg Se #r Ala Ser Gly Ala Val             980       #           985       #           990 Phe Arg Ser Lys Lys Val Val Val Ser Leu Pr #o Thr Thr Leu Tyr Pro         995           #       1000           #      1005 Thr Leu Thr Phe Ser Pro Pro Leu Pro Ala Gl #u Lys Gln Ala Leu Ala     1010              #   1015               #  1020 Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Lys Il #e Val Phe Val Trp Asp 1025                1030 #                1035  #               1040 Lys Pro Trp Trp Arg Glu Gln Gly Phe Ser Gl #y Val Leu Gln Ser Ser                 1045  #               1050   #              1055 Cys Asp Pro Ile Ser Phe Ala Arg Asp Thr Se #r Ile Asp Val Asp Arg             1060      #           1065       #          1070 Gln Trp Ser Ile Thr Cys Phe Met Val Gly As #p Pro Gly Arg Lys Trp         1075          #       1080           #      1085 Ser Gln Gln Ser Lys Gln Val Arg Gln Lys Se #r Val Trp Asp Gln Leu     1090              #   1095               #  1100 Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gln Va #l Pro Glu Pro Ala Asn 1105                1110 #                1115  #               1120 Val Leu Glu Ile Glu Trp Ser Lys Gln Gln Ty #r Phe Gln Gly Ala Pro                 1125  #               1130   #              1135 Ser Ala Val Tyr Gly Leu Asn Asp Leu Ile Th #r Leu Gly Ser Ala Leu             1140      #           1145       #          1150 Arg Thr Pro Phe Lys Ser Val His Phe Val Gl #y Thr Glu Thr Ser Leu         1155          #       1160           #      1165 Val Trp Lys Gly Tyr Met Glu Gly Ala Ile Ar #g Ser Gly Gln Arg Gly     1170              #   1175               #  1180 Ala Ala Glu Val Val Ala Ser Leu Val Pro Al #a Ala 1185                1190 #                1195 <210> SEQ ID NO 32 <211> LENGTH: 2490 <212> TYPE: DNA <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION: GST:glyc(-)APAO open reading # frame, 2490 nt;       GST and linker, nt 1-687; Glyc  #(-) APAO, nt 688-2490; mutation in       putative glycosylation sites in bold # and underlined, nt  1288-1290       (AAT-> TCC) and nt 1303-1305 (A #GC-> AAC). <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)...(2487) <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(687) <223> OTHER INFORMATION: GST and linker <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (688)...(2490) <223> OTHER INFORMATION: Glyc (-) APAO <220> FEATURE: <221> NAME/KEY: mutation <222> LOCATION: (1288)...(1290) <223> OTHER INFORMATION: mutation in putative glyc #osylation site       (AAT->TCC) <220> FEATURE: <221> NAME/KEY: mutation <222> LOCATION: (1303)...(1305) <223> OTHER INFORMATION: mutation in putative glyc #osylation site       (AGC->AAC) <400> SEQUENCE: 32 atg tcc cct ata cta ggt tat tgg aaa att aa #g ggc ctt gtg caa ccc       48 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Ly #s Gly Leu Val Gln Pro  1               5   #                 10  #                 15 act cga ctt ctt ttg gaa tat ctt gaa gaa aa #a tat gaa gag cat ttg       96 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Ly #s Tyr Glu Glu His Leu              20      #             25      #             30 tat gag cgc gat gaa ggt gat aaa tgg cga aa #c aaa aag ttt gaa ttg      144 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg As #n Lys Lys Phe Glu Leu          35          #         40          #         45 ggt ttg gag ttt ccc aat ctt cct tat tat at #t gat ggt gat gtt aaa      192 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Il #e Asp Gly Asp Val Lys      50              #     55              #     60 tta aca cag tct atg gcc atc ata cgt tat at #a gct gac aag cac aac      240 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Il #e Ala Asp Lys His Asn  65                  # 70                  # 75                  # 80 atg ttg ggt ggt tgt cca aaa gag cgt gca ga #g att tca atg ctt gaa      288 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Gl #u Ile Ser Met Leu Glu                  85  #                 90  #                 95 gga gcg gtt ttg gat att aga tac ggt gtt tc #g aga att gca tat agt      336 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Se #r Arg Ile Ala Tyr Ser             100       #           105       #           110 aaa gac ttt gaa act ctc aaa gtt gat ttt ct #t agc aag cta cct gaa      384 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Le #u Ser Lys Leu Pro Glu         115           #       120           #       125 atg ctg aaa atg ttc gaa gat cgt tta tgt ca #t aaa aca tat tta aat      432 Met Leu Lys Met Phe Glu Asp Arg Leu Cys Hi #s Lys Thr Tyr Leu Asn     130               #   135               #   140 ggt gat cat gta acc cat cct gac ttc atg tt #g tat gac gct ctt gat      480 Gly Asp His Val Thr His Pro Asp Phe Met Le #u Tyr Asp Ala Leu Asp 145                 1 #50                 1 #55                 1 #60 gtt gtt tta tac atg gac cca atg tgc ctg ga #t gcg ttc cca aaa tta      528 Val Val Leu Tyr Met Asp Pro Met Cys Leu As #p Ala Phe Pro Lys Leu                 165   #               170   #               175 gtt tgt ttt aaa aaa cgt att gaa gct atc cc #a caa att gat aag tac      576 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pr #o Gln Ile Asp Lys Tyr             180       #           185       #           190 ttg aaa tcc agc aag tat ata gca tgg cct tt #g cag ggc tgg caa gcc      624 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Le #u Gln Gly Trp Gln Ala         195           #       200           #       205 acg ttt ggt ggt ggc gac cat cct cca aaa tc #g gat ctg gtt ccg cgt      672 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Se #r Asp Leu Val Pro Arg     210               #   215               #   220 gga tcc ccg gaa ttc atg gca ctt gca ccg ag #c tac atc aat ccc cca      720 Gly Ser Pro Glu Phe Met Ala Leu Ala Pro Se #r Tyr Ile Asn Pro Pro 225                 2 #30                 2 #35                 2 #40 aac gtc gcc tcc cca gca ggg tat tct cac gt #c ggc gta ggc cca gac      768 Asn Val Ala Ser Pro Ala Gly Tyr Ser His Va #l Gly Val Gly Pro Asp                 245   #               250   #               255 gga ggg agg tat gtg aca ata gct gga cag at #t gga caa gac gct tcg      816 Gly Gly Arg Tyr Val Thr Ile Ala Gly Gln Il #e Gly Gln Asp Ala Ser             260       #           265       #           270 ggc gtg aca gac cct gcc tac gag aaa cag gt #t gcc caa gca ttc gcc      864 Gly Val Thr Asp Pro Ala Tyr Glu Lys Gln Va #l Ala Gln Ala Phe Ala         275           #       280           #       285 aat ctg cga gct tgc ctt gct gca gtt gga gc #c act tca aac gac gtc      912 Asn Leu Arg Ala Cys Leu Ala Ala Val Gly Al #a Thr Ser Asn Asp Val     290               #   295               #   300 acc aag ctc aat tac tac atc gtc gac tac gc #c ccg agc aaa ctc acc      960 Thr Lys Leu Asn Tyr Tyr Ile Val Asp Tyr Al #a Pro Ser Lys Leu Thr 305                 3 #10                 3 #15                 3 #20 gca att gga gat ggg ctg aag gct acc ttt gc #c ctt gac agg ctc cct     1008 Ala Ile Gly Asp Gly Leu Lys Ala Thr Phe Al #a Leu Asp Arg Leu Pro                 325   #               330   #               335 cct tgc acg ctg gtg cca gtg tcg gcc ttg tc #t tca cct gaa tac ctc     1056 Pro Cys Thr Leu Val Pro Val Ser Ala Leu Se #r Ser Pro Glu Tyr Leu             340       #           345       #           350 ttt gag gtt gat gcc acg gcg ctg gtg ccg gg #a cac acg acc cca gac     1104 Phe Glu Val Asp Ala Thr Ala Leu Val Pro Gl #y His Thr Thr Pro Asp         355           #       360           #       365 aac gtt gcg gac gtg gta gtg gtg ggc gct gg #c ttg agc ggt ttg gag     1152 Asn Val Ala Asp Val Val Val Val Gly Ala Gl #y Leu Ser Gly Leu Glu     370               #   375               #   380 acg gca cgc aaa gtc cag gcc gcc ggt ctg tc #c tgc ctc gtt ctt gag     1200 Thr Ala Arg Lys Val Gln Ala Ala Gly Leu Se #r Cys Leu Val Leu Glu 385                 3 #90                 3 #95                 4 #00 gcg atg gat cgt gta ggg gga aag act ctg ag #c gta caa tcg ggt ccc     1248 Ala Met Asp Arg Val Gly Gly Lys Thr Leu Se #r Val Gln Ser Gly Pro                 405   #               410   #               415 ggc agg acg act atc aac gac ctc ggc gct gc #g tgg atc tcc gac agc     1296 Gly Arg Thr Thr Ile Asn Asp Leu Gly Ala Al #a Trp Ile Ser Asp Ser             420       #           425       #           430 aac caa aac gaa gta tcc aga ttg ttt gaa ag #a ttt cat ttg gag ggc     1344 Asn Gln Asn Glu Val Ser Arg Leu Phe Glu Ar #g Phe His Leu Glu Gly         435           #       440           #       445 gag ctc cag agg acg act gga aat tca atc ca #t caa gca caa gac ggt     1392 Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile Hi #s Gln Ala Gln Asp Gly     450               #   455               #   460 aca acc act aca gct cct tat ggt gac tcc tt #g ctg agc gag gag gtt     1440 Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Le #u Leu Ser Glu Glu Val 465                 4 #70                 4 #75                 4 #80 gca agt gca ctt gcg gaa ctc ctc ccc gta tg #g tct cag ctg atc gaa     1488 Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Tr #p Ser Gln Leu Ile Glu                 485   #               490   #               495 gag cat agc ctt caa gac ctc aag gcg agc cc #t cag gcg aag cgg ctc     1536 Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pr #o Gln Ala Lys Arg Leu             500       #           505       #           510 gac agt gtg agc ttc gcg cac tac tgt gag aa #g gaa cta aac ttg cct     1584 Asp Ser Val Ser Phe Ala His Tyr Cys Glu Ly #s Glu Leu Asn Leu Pro         515           #       520           #       525 gct gtt ctc ggc gta gca aac cag atc aca cg #c gct ctg ctc ggt gtg     1632 Ala Val Leu Gly Val Ala Asn Gln Ile Thr Ar #g Ala Leu Leu Gly Val     530               #   535               #   540 gaa gcc cac gag atc agc atg ctt ttt ctc ac #c gac tac atc aag agt     1680 Glu Ala His Glu Ile Ser Met Leu Phe Leu Th #r Asp Tyr Ile Lys Ser 545                 5 #50                 5 #55                 5 #60 gcc acc ggt ctc agt aat att ttc tcg gac aa #g aaa gac ggc ggg cag     1728 Ala Thr Gly Leu Ser Asn Ile Phe Ser Asp Ly #s Lys Asp Gly Gly Gln                 565   #               570   #               575 tat atg cga tgc aaa aca ggt atg cag tcg at #t tgc cat gcc atg tca     1776 Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Il #e Cys His Ala Met Ser             580       #           585       #           590 aag gaa ctt gtt cca ggc tca gtg cac ctc aa #c acc ccc gtc gct gaa     1824 Lys Glu Leu Val Pro Gly Ser Val His Leu As #n Thr Pro Val Ala Glu         595           #       600           #       605 att gag cag tcg gca tcc ggc tgt aca gta cg #a tcg gcc tcg ggc gcc     1872 Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Ar #g Ser Ala Ser Gly Ala     610               #   615               #   620 gtg ttc cga agc aaa aag gtg gtg gtt tcg tt #a ccg aca acc ttg tat     1920 Val Phe Arg Ser Lys Lys Val Val Val Ser Le #u Pro Thr Thr Leu Tyr 625                 6 #30                 6 #35                 6 #40 ccc acc ttg aca ttt tca cca cct ctt ccc gc #c gag aag caa gca ttg     1968 Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Al #a Glu Lys Gln Ala Leu                 645   #               650   #               655 gcg gaa aat tct atc ctg ggc tac tat agc aa #g ata gtc ttc gta tgg     2016 Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Ly #s Ile Val Phe Val Trp             660       #           665       #           670 gac aag ccg tgg tgg cgc gaa caa ggc ttc tc #g ggc gtc ctc caa tcg     2064 Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Se #r Gly Val Leu Gln Ser         675           #       680           #       685 agc tgt gac ccc atc tca ttt gcc aga gat ac #c agc atc gac gtc gat     2112 Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Th #r Ser Ile Asp Val Asp     690               #   695               #   700 cga caa tgg tcc att acc tgt ttc atg gtc gg #a gac ccg gga cgg aag     2160 Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gl #y Asp Pro Gly Arg Lys 705                 7 #10                 7 #15                 7 #20 tgg tcc caa cag tcc aag cag gta cga caa aa #g tct gtc tgg gac caa     2208 Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Ly #s Ser Val Trp Asp Gln                 725   #               730   #               735 ctc cgc gca gcc tac gag aac gcc ggg gcc ca #a gtc cca gag ccg gcc     2256 Leu Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gl #n Val Pro Glu Pro Ala             740       #           745       #           750 aac gtg ctc gaa atc gag tgg tcg aag cag ca #g tat ttc caa gga gct     2304 Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gl #n Tyr Phe Gln Gly Ala         755           #       760           #       765 ccg agc gcc gtc tat ggg ctg aac gat ctc at #c aca ctg ggt tcg gcg     2352 Pro Ser Ala Val Tyr Gly Leu Asn Asp Leu Il #e Thr Leu Gly Ser Ala     770               #   775               #   780 ctc aga acg ccg ttc aag agt gtt cat ttc gt #t gga acg gag acg tct     2400 Leu Arg Thr Pro Phe Lys Ser Val His Phe Va #l Gly Thr Glu Thr Ser 785                 7 #90                 7 #95                 8 #00 tta gtt tgg aaa ggg tat atg gaa ggg gcc at #a cga tcg ggt caa cga     2448 Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Il #e Arg Ser Gly Gln Arg                 805   #               810   #               815 ggt gct gca gaa gtt gtg gct agc ctg gtg cc #a gca gca tag              #2490 Gly Ala Ala Glu Val Val Ala Ser Leu Val Pr #o Ala Ala             820       #           825 <210> SEQ ID NO 33 <211> LENGTH: 829 <212> TYPE: PRT <213> ORGANISM: Unknown <400> SEQUENCE: 33 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Ly #s Gly Leu Val Gln Pro  1               5   #                10   #                15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Ly #s Tyr Glu Glu His Leu             20       #            25       #            30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg As #n Lys Lys Phe Glu Leu         35           #        40           #        45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Il #e Asp Gly Asp Val Lys     50               #    55               #    60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Il #e Ala Asp Lys His Asn 65                   #70                   #75                   #80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Gl #u Ile Ser Met Leu Glu                 85   #                90   #                95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Se #r Arg Ile Ala Tyr Ser             100       #           105       #           110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Le #u Ser Lys Leu Pro Glu         115           #       120           #       125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys Hi #s Lys Thr Tyr Leu Asn     130               #   135               #   140 Gly Asp His Val Thr His Pro Asp Phe Met Le #u Tyr Asp Ala Leu Asp 145                 1 #50                 1 #55                 1 #60 Val Val Leu Tyr Met Asp Pro Met Cys Leu As #p Ala Phe Pro Lys Leu                 165   #               170   #               175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pr #o Gln Ile Asp Lys Tyr             180       #           185       #           190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Le #u Gln Gly Trp Gln Ala         195           #       200           #       205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Se #r Asp Leu Val Pro Arg     210               #   215               #   220 Gly Ser Pro Glu Phe Met Ala Leu Ala Pro Se #r Tyr Ile Asn Pro Pro 225                 2 #30                 2 #35                 2 #40 Asn Val Ala Ser Pro Ala Gly Tyr Ser His Va #l Gly Val Gly Pro Asp                 245   #               250   #               255 Gly Gly Arg Tyr Val Thr Ile Ala Gly Gln Il #e Gly Gln Asp Ala Ser             260       #           265       #           270 Gly Val Thr Asp Pro Ala Tyr Glu Lys Gln Va #l Ala Gln Ala Phe Ala         275           #       280           #       285 Asn Leu Arg Ala Cys Leu Ala Ala Val Gly Al #a Thr Ser Asn Asp Val     290               #   295               #   300 Thr Lys Leu Asn Tyr Tyr Ile Val Asp Tyr Al #a Pro Ser Lys Leu Thr 305                 3 #10                 3 #15                 3 #20 Ala Ile Gly Asp Gly Leu Lys Ala Thr Phe Al #a Leu Asp Arg Leu Pro                 325   #               330   #               335 Pro Cys Thr Leu Val Pro Val Ser Ala Leu Se #r Ser Pro Glu Tyr Leu             340       #           345       #           350 Phe Glu Val Asp Ala Thr Ala Leu Val Pro Gl #y His Thr Thr Pro Asp         355           #       360           #       365 Asn Val Ala Asp Val Val Val Val Gly Ala Gl #y Leu Ser Gly Leu Glu     370               #   375               #   380 Thr Ala Arg Lys Val Gln Ala Ala Gly Leu Se #r Cys Leu Val Leu Glu 385                 3 #90                 3 #95                 4 #00 Ala Met Asp Arg Val Gly Gly Lys Thr Leu Se #r Val Gln Ser Gly Pro                 405   #               410   #               415 Gly Arg Thr Thr Ile Asn Asp Leu Gly Ala Al #a Trp Ile Ser Asp Ser             420       #           425       #           430 Asn Gln Asn Glu Val Ser Arg Leu Phe Glu Ar #g Phe His Leu Glu Gly         435           #       440           #       445 Glu Leu Gln Arg Thr Thr Gly Asn Ser Ile Hi #s Gln Ala Gln Asp Gly     450               #   455               #   460 Thr Thr Thr Thr Ala Pro Tyr Gly Asp Ser Le #u Leu Ser Glu Glu Val 465                 4 #70                 4 #75                 4 #80 Ala Ser Ala Leu Ala Glu Leu Leu Pro Val Tr #p Ser Gln Leu Ile Glu                 485   #               490   #               495 Glu His Ser Leu Gln Asp Leu Lys Ala Ser Pr #o Gln Ala Lys Arg Leu             500       #           505       #           510 Asp Ser Val Ser Phe Ala His Tyr Cys Glu Ly #s Glu Leu Asn Leu Pro         515           #       520           #       525 Ala Val Leu Gly Val Ala Asn Gln Ile Thr Ar #g Ala Leu Leu Gly Val     530               #   535               #   540 Glu Ala His Glu Ile Ser Met Leu Phe Leu Th #r Asp Tyr Ile Lys Ser 545                 5 #50                 5 #55                 5 #60 Ala Thr Gly Leu Ser Asn Ile Phe Ser Asp Ly #s Lys Asp Gly Gly Gln                 565   #               570   #               575 Tyr Met Arg Cys Lys Thr Gly Met Gln Ser Il #e Cys His Ala Met Ser             580       #           585       #           590 Lys Glu Leu Val Pro Gly Ser Val His Leu As #n Thr Pro Val Ala Glu         595           #       600           #       605 Ile Glu Gln Ser Ala Ser Gly Cys Thr Val Ar #g Ser Ala Ser Gly Ala     610               #   615               #   620 Val Phe Arg Ser Lys Lys Val Val Val Ser Le #u Pro Thr Thr Leu Tyr 625                 6 #30                 6 #35                 6 #40 Pro Thr Leu Thr Phe Ser Pro Pro Leu Pro Al #a Glu Lys Gln Ala Leu                 645   #               650   #               655 Ala Glu Asn Ser Ile Leu Gly Tyr Tyr Ser Ly #s Ile Val Phe Val Trp             660       #           665       #           670 Asp Lys Pro Trp Trp Arg Glu Gln Gly Phe Se #r Gly Val Leu Gln Ser         675           #       680           #       685 Ser Cys Asp Pro Ile Ser Phe Ala Arg Asp Th #r Ser Ile Asp Val Asp     690               #   695               #   700 Arg Gln Trp Ser Ile Thr Cys Phe Met Val Gl #y Asp Pro Gly Arg Lys 705                 7 #10                 7 #15                 7 #20 Trp Ser Gln Gln Ser Lys Gln Val Arg Gln Ly #s Ser Val Trp Asp Gln                 725   #               730   #               735 Leu Arg Ala Ala Tyr Glu Asn Ala Gly Ala Gl #n Val Pro Glu Pro Ala             740       #           745       #           750 Asn Val Leu Glu Ile Glu Trp Ser Lys Gln Gl #n Tyr Phe Gln Gly Ala         755           #       760           #       765 Pro Ser Ala Val Tyr Gly Leu Asn Asp Leu Il #e Thr Leu Gly Ser Ala     770               #   775               #   780 Leu Arg Thr Pro Phe Lys Ser Val His Phe Va #l Gly Thr Glu Thr Ser 785                 7 #90                 7 #95                 8 #00 Leu Val Trp Lys Gly Tyr Met Glu Gly Ala Il #e Arg Ser Gly Gln Arg                 805   #               810   #               815 Gly Ala Ala Glu Val Val Ala Ser Leu Val Pr #o Ala Ala             820       #           825 

What is claimed is:
 1. An isolated polynucleotide comprising a member selected from: a) a polynucleotide that encodes a polypeptide selected from SEQ ID NO: 6, SEQ ID NO: 11, and SEQ ID NO: 23; and b) a polynucleotide comprising a polynucleotide selected from SEQ ID NO: 5, SEQ ID NO: 10, and SEQ ID NO:
 22. 2. A recombinant expression cassette comprising a member of claim
 1. 3. A vector comprising the recombinant expression cassette of claim
 2. 4. A host cell comprising the recombinant expression cassette of claim
 2. 5. The host cell of claim 4 wherein the cell is a plant cell.
 6. A transformed plant cell comprising the polynucleotide of claim
 1. 7. The transformed plant cell of claim 6 wherein the plant cell is selected from the group consisting of maize, sorghum, wheat, tomato, soybean, alfalfa, sunflower, canola, cotton, and rice.
 8. A plant comprising the polynucleotide of claim
 1. 9. A seed from the plant of claim
 8. 10. An isolated polynucleotide comprising the polynucleotide of claim 1 fused to a plant signal sequence.
 11. The polynucleotide of claim 10 wherein the plant signal sequence is apoplast targeting sequence.
 12. A method of reducing pathogenicity of a fungus producing fumonisin or a structurally related mycotoxin, comprising: a) transforming a plant cell with a vector comprising the polynucleotide of claim 1 operably linked to a promoter; b) growing the plant cell under plant growing conditions; and c) inducing expression of said polynucleotides for a time sufficient for amounts of the fumonisin esterase and APAO enzymes to accumulate to levels that can inhibit the fungus.
 13. A method of making an APAO enzyme comprising the steps of: a) expressing the nucleic acid of claim 1 in a recombinantly engineered cell; and b) purifying the enzyme.
 14. A method of making an APAO enzyme comprising the steps of: a) expressing the nucleic acid of claim 1 in a plant; and b) purifying the enzyme from the plant seed or other plant parts. 